Methods of treating inflammatory and autoimmune diseases with natalizumab

ABSTRACT

Natalizumab is a safe and efficacious treatment for inflammatory and autoimmune diseases, such as multiple sclerosis, Crohn&#39;s Disease, and rheumatoid arthritis. Chain swapping between natalizumab and IgG4 molecules acts to reduce the level of bivalent natalizumab present following administration of natalizumab, and thus to lower the activity of natalizumab in the patient. Differences in IgG4 levels across patients or within a single patient across time may change the pharmacokinetic profile of natalizumab. Patients with lower levels of IgG4 may experience higher nadir levels of natalizumab during a dosing period. Monitoring IgG4 and/or bivalent natalizumab levels, and determining a dose or dosage period based on the monitoring may improve the safety and/or efficacy of natalizumab therapy.

PRIORITY CLAIM

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/779,190, filed Mar. 3, 2006, the entire contents of which arehereby incorporated herein.

TECHNICAL FIELD

The invention relates to methods of treating inflammatory and autoimmunediseases with a recombinant antibody. These methods improve the safetyof treatment by adjusting the dose based on IgG4 antibodies in thepatient.

BACKGROUND ART

The migration of lymphocytes from the peripheral blood across the bloodbrain barrier has been reported to initiate the development of severalcentral nervous system (CNS) inflammatory diseases. Lymphocyte entryinto the CNS is mediated by cellular adhesion molecules (O'Neill et al.,Immunology 72:520-525 (1991); Raine et al., Lab. Invest. 63:476-489(1990); Yednock et al., Nature 356:63-66 (1992); Baron et al., J. Exp.Med. 177:57-68 (1993); Steffen et al., Am. J. Path. 145:189-201 (1994);Christensen et al., J. Immunol. 154:5293-5301 (1995)).

Cellular adhesion molecules present on the cell surface mediate thedirect binding of one cell to another (Long et al., Exp. Hematol.20:288-301 (1992)). The integrin and immunoglobulin supergene familiesof adhesion molecules regulate lymphocyte traffic into the CNS (Hemleret al., Annu. Rev. Immunol. 8:365-400 (1990); Springer et al., Cell76:301-314 (1994); Issekutz et al., Curr. Opin. Immunol. 4:287-293(1992)). Adhesion molecules have been widely reported to mediateinflammatory and autoimmune diseases, for example, asthma, Alzheimer'sdisease, atherosclerosis, AIDS dementia, diabetes, inflammatory boweldisease, multiple sclerosis, rheumatoid arthritis, tissuetransplantation rejection, and tumor metastasis.

Integrins are heterodimers of non-covalently linked α and β chains(Hemler et al., Annu. Rev. Immunol. 8:365-400 (1990)). The α4β1 (alsocalled very late activation antigen-4 VLA-4) and α4β7 integrins arepresent on the surface of most types of white blood cells, where theymediate white cell binding to endothelial cells by interacting withtheir cognate receptors, vascular cell adhesion molecule-1 (VCAM-1) andmucosal addressin cellular adhesion molecule-1 (MAdCAM-1), on theendothelial cell surface. Integrins are believed to play an importantrole in immune cell adhesion to the endothelial cell layer on bloodvessels, facilitating their subsequent migration into inflamed tissues.Several studies implicate VLA-4 and, in particular the α4 integrinsubunit, in CNS inflammation (Yednock et al., Nature 356:63-66 (1992);Baron et al., J. Exp. Med. 177:57-68 (1993); Steffen et al., Am. J.Path. 145:189-201 (1994); Christensen et al., J. Immunol. 154:5293-5301(1995). It has also been reported that VCAM-1 expression is elevated ininflamed brain tissue relative to normal brain tissue (Cannella andRaine, Ann. Neurol. 37:424-435 (1995); Washington et al., Ann. Neurol.35:89-97 (1994); Dore-Duffy et al., Frontiers in Cerebral VascularBiology: Transport and Its Regulation, 243-248 (Eds. Drewes & Betz,Plenum, N.Y. 1993)).

The interaction between α4β1 and its targets is a component of theinflammation that takes place in the CNS of patients with multiplesclerosis (MS). Under normal conditions, VCAM-1 is not expressed in thebrain parenchyma. However, in the presence of pro-inflammatorycytokines, VCAM-1 is upregulated on endothelial cells and on microglialcells near the sites of inflammation (Elices et al., Cell 60:577-584(1990); Lobb and Hemler, J. Clin. Invest. 94:1722-1728 (1994); Petersonet al., J. Neuropathy Exp. Neurol. 61:539-546 (2002)). Further,osteopontin, which exhibits many properties of a proinflammatorycytokine, is also upregulated in MS lesions (Chabas et al., Science294:1731-1735 (2001)).

MS is a serious and disabling inflammatory and autoimmune disease ofyoung adults, with a peak age of onset in the third decade of life. Mostindividuals present with the relapsing-remitting form of the disease andexperience recurrent attacks, which, over time, result in accumulatingpermanent physical disability and cognitive decline. About 70% of theseindividuals will eventually enter a phase of progressive neurologicaldecline (secondary progressive MS), with or without superimposedrelapses. Current treatments are minimally effective for secondaryprogressive MS. The majority of patients suffer permanent neurologicaldysfunction and, on average, have a life expectancy of six to sevenyears after the onset of disease.

Currently, four therapies are approved in the United States for thetreatment of relapsing forms of MS. The interferons, Betaseron®(interferon β-1b SC (subcutaneous)), AVONEX® (interferon β-1a IM(intramuscular)), and Rebif® (interferon β-1a SC), are cytokines withantiviral, antiproliferative, and immunomodulatory activities. Copaxone®(glatiramer acetate) is a mixture of synthetic polypeptides with apoorly understood mechanism of action. The β-interferons can produceserious adverse events and some evidence suggests that copaxone isineffective (Munari, et al., The Cochrane Library, Issue 1, Chichester,UK: John Wiley & Sons, Ltd. (2004)).

Serious adverse events of β-interferons include rare reports ofhypersensitivity reactions, depression and suicide, decreased peripheralblood counts, hepatic injury, cardiomyopathy, and various autoimmunedisorders (Betaseron Package Insert, 2003; Rebif Package Insert, 2004;AVONEX® Package Insert, 2005). The development of neutralizingantibodies to interferons is associated with a loss of efficacy.Antibodies that develop to a β-interferon cross-react with otherinterferons leading to loss of efficacy for the entire class in suchpatients (IFNB MS Study Group, Neurology 47:889-894 (1996); PRISMS StudyGroup, Neurology 56:1628-1636 (2001); Kappos et al., Neurology 65:40-47(2005)). As a result, in the United States alone, over 50,000 patientswho were previously treated no longer receive therapy. Thus, there is alarge group of patients with active MS who are currently not receivingany approved therapy.

Among those patients who do receive treatment, a significant numbercontinue to experience disease activity, as observed clinically and bymagnetic resonance imaging (MM). Although a variety of therapeuticstrategies are currently used in clinical practice to managebreakthrough disease while on treatment (e.g., switching therapy,changing dose and frequency of interferon, combination therapy), thesimilar efficacy between available medications and lack of clinical datademonstrating the effectiveness of any of these strategies inbreakthrough patients makes the decision of what to do for thesepatients largely empirical. Each of the partially effective approvedmedications leads to an approximately 30% reduction in relapse rate andlimited impact on disability progression (IFNB MS Study Group, Neurology43:655-661 (1993); Jacobs et al., Ann. Neurol. 39:285-289 (1996); PRISMSStudy Group, Lancet 352:1498-1504 (1998)); Johnson et al., Neurology45:1268-1276 (1995)). Data from the Phase 3 trials of β-interferon in MSshow that 62% to 75% of subjects experienced at least one relapse duringthese 2-year trials despite interferon treatment (IFNB MS Study Group,Neurology 43:655-661 (1993); Jacobs et al., Ann. Neurol. 39:285-289(1996); PRISMS Study Group, Lancet 352:1498-1504 (1998)). Similarly, 66%of subjects in the Phase 3 MS trial of glatiramer acetate experienced atleast one relapse during the 2-year period, a proportion that was notsignificantly different from placebo (Johnson et al., Neurology45:1268-1276 (1995)).

Progressive Multifocal Leukoencephalopathy (PML) is a severe, rapidlyprogressive disease that destroys the myelin coating which protectsnerve cells. PML occurs almost exclusively in severely immunosuppressedpatients and is frequently associated with lymphoproliferative and otherchronic diseases, such as AIDS, Hodgkin's disease, chronic lymphocyticleukemia, sarcoidosis, tuberculosis, systemic lupis erythematosis, andorgan transplantation. JC virus (JCV) is the etiological agent of PMLand may result from a primary infection or follow reactivation of latentvirus.

Natalizumab, an α4-integrin antagonist, has been used successfully totreat diseases with inflammatory and/or autoimmune components, forexample, MS, Crohn's Disease, and rheumatoid arthritis. Natalizumab is ahumanized IgG₄κ monoclonal antibody directed against the α4-integrinsα4β1 and α4β7. Chain swapping between natalizumab and other IgG₄molecules may affect the pharmacokinetics of natalizumab. Differences inthe concentration of IgG₄ between patients or within a patient over timemay lead to differences in the concentration of bivalent natalizumabdelivered over a dosing period. This may lead to variation in safetyand/or efficacy between patients or within a patient over successivedosage periods.

Variation in IgG₄ levels may also lead to excessive natalizumab activityin certain patients. This may lead to an increased risk of infection inthose pateints. For example, there are three known cases of PMLoccurring during or after administration of natalizumab, two provedfatal and one patient recovered. All three cases occurred in patients onconcomitant medications which may have contributed to immunosuppression.

Thus, there is a need in the art for determining the relationshipbetween IgG₄ levels and natalizumab pharmacokinetics, and for adjustingnatalizumab dose and dosage interval in certain patients in view of thisinformation to improve the safety and/or efficacy of natalizumabtreatment.

SUMMARY

The invention provides safer methods of using natalizumab to treatpatients with inflammatory and autoimmune diseases.

In a first aspect, the invention provides a method of treating a patientwith an inflammatory or autoimmune disease with natalizumab byadministering a dose of natalizumab for a first dosage period;monitoring the amount of bivalent natalizumab in the patient's plasma orserum during the first dosage period; determining a second dose ofnatalizumab based on the level of bivalent natalizumab observed; andadministering a second dose of natalizumab for a second dosage period;wherein the second dose improves the safety and/or efficacy of thetreatment during the second dosage period. In an embodiment of themethod the monitoring shows that the amount of bivalent natalizumab inthe patient's plasma or serum remains above a predetermined level duringthe first dosage period, and the corrected dose of natalizumabadministered over the second dosage period is designed to achieve areduction of the natalizumab level during the second dosage period tobelow the predetermined level during at least a portion of the seconddosage period. In an embodiment the second dose is lower than the firstdose. In an embodiment the second dosage period is longer than the firstdosage period. In an embodiment the corrected dose is lower than thefirst dose, and wherein the second dosage period is longer than thefirst dosage period. In an embodiment the first dose is 300 mgadministered by IV infusion and the first dosage period is four weeks.In an embodiment the predetermined level is about 1 μg/ml, and whereinthe second dose is less than 300 mg administered by IV infusion and thesecond dosage period is more than four weeks. In an embodiment thepredetermined level is about 0.5 μg/ml, and wherein the second dose isless than 300 mg administered by IV infusion and the second dosageperiod is more than four weeks. In an embodiment the predetermined levelis about 0.1 μg/ml, and wherein the second dose is less than 300 mgadministered by IV infusion and the second dosage period is more thanfour weeks.

The invention also provides a method of treating a patient with aninflammatory or autoimmune disease with natalizumab, wherein the amountof bivalent natalizumab in the patient's plasma or serum falls below apredetermined level during the first dosage period within apredetermined time after administration of the first dose, and whereinthe second dose of natalizumab administered over the second dosageperiod is designed to maintain the natalizumab level above thepredetermined level.

In an embodiment of the method the disease is multiple sclerosis. In anembodiment the multiple sclerosis is selected from relapsing remitting,secondary progressive, primary progressive, and chronic progressivemultiple sclerosis. In an embodiment of the method the disease isinflammatory bowel disease or rheumatoid arthritis. In an embodiment theinflammatory bowel disease is Crohn's Disease.

In an embodiment the method further includes monitoring the patient forindicators of serious infection and/or treating the patient withprophylaxis designed to reduce the risk of developing serious infection.

In an embodiment the method further includes monitoring the patient forindicators of progressive multifocal leukoencephalopathy. In anembodiment the monitoring detects JCV in the patient's urine, blood,and/or cerebrospinal fluid. In an embodiment the monitoring comprisesserially removing samples of the patient's blood, measuring the amountof IgG antibodies to JCV in the samples, and comparing the amount of theantibodies in the samples. In an embodiment the monitoring furthercomprises measuring the amount of IgM antibodies to JCV in the samples,and comparing the amount of the IgM and IgG antibodies in the samples.In an embodiment the monitoring detects seroconversion and/or anincreasing titer of JCV in the patient's urine and/or blood, and furtherincludes removing a sample of the patient's cerebrospinal fluid when thecomparison of the serial urine and/or blood samples detectseroconversion and/or an increasing titer of JCV; and testing thecerebrospinal fluid for the presence of JCV. In an embodiment themonitoring includes testing for clinical and/or radiologic symptoms ofprogressive multifocal leukoencephalopathy. In an embodiment the testingfor clinical symptoms includes testing for new or worsening neurologicalsymptoms. In an embodiment the neurological symptoms include one or moreof central blindness, mental confusion, personality change, anddyskinesia. In an embodiment the testing for radiologic symptomsincludes performing a Gd-enhanced magnetic resonance imaging scan. In anembodiment the method includes, in the presence of indicators ofprogressive multifocal leukoencephalopathy, providing at least onetreatment selected from intravenous immunoglobulin therapy,plasmapheresis, and antiviral therapy. In an embodiment the antiviraltherapy comprises the administration of at least one therapeuticallyeffective dose of an antiviral agent selected from cytosine arabinoside(cytarabine), cidofovir, and a serotonin antagonist. In an embodimentthe serotonin antagonist is a 5HT2a antagonist.

In an embodiment of the method the patient is not treated simultaneouslywith natalizumab and an immunosuppressive or antineoplastic agent. In anembodiment the immunosuppressive or antineoplastic agent is selectedfrom one or more of chlorambucil, melphalan, 6-mercaptopurine, thiotepa,ifodfamide, dacarbazine, procarbazine, temozolomide, hexamethylmelamine,doxorubicine, daunarubicine, idarubicin, epirubicin, irinotecan,methotrexate, etoposide, vincristine, vinblastine, vinorelbine,cytarabine, busulfan, amonifide, 5-fluorouracil, topotecan, mustargen,bleomycin, lomustine, semustine, mitomycin C, mutamycin, cisplatin,carboplatin, oxaliplatin, methotrexate, trimetrexate, raltitrexid,flurorodeoxyuridine, capecitabine, ftorafur, 5-ethynyluracil,6-thioguanine, cladribine, pentostatin, teniposide, mitoxantrone,losoxantrone, actinomycin D, vindesine, docetaxel, amifostine,interferon alpha, tamoxefen, medroxyprogesterone, megestrol, raloxifene,letrozole, anastrzole, flutamide, bicalutamide, retinoic acids, arsenictrioxide, rituximab, CAMPATH-1, mylotarg, mycophenolic acid, tacrolimus,glucocorticoids, sulfasalazine, glatiramer, fumarate, laquinimod,FTY-720, interferon tau, daclizumab, infliximab, IL10, anti-IL2 receptorantibody, anti-IL-12 antibody, anti-IL6 receptor antibody, CDP-571,adalimumab, entaneracept, leflunomide, anti-interferon gamma antibody,abatacept, fludarabine, cyclophosphamide, azathioprine, cyclosporine,intravenous immunoglobulin, 5-ASA (mesalamine), and a β-interferon.

In another aspect the invention provides a method of treating a patientwith an inflammatory or autoimmune disease with natalizumab bydetermining the amount of IgG4 in the patient's plasma or serum;determining a dose and dosage period of natalizumab based on the amountof IgG4 in the patient's plasma or serum; and administering the dose ofnatalizumab to the patient over the dosage period; wherein the dose anddosage period improve the safety and/or efficacy of the treatmentcompared to the safety and/or efficacy provided by the standard dose anddosage period of natalizumab. In an embodiment of the method thestandard dose is 300 mg by IV infusion and the standard dosage period isevery four weeks. In an embodiment the amount of IgG4 in the patient'sblood is below 200 μg/ml and the determined dose of natalizumab is below300 mg by IV infusion. In an embodiment the amount of IgG4 in thepatient's blood is below 200 ug/ml and the determined dosage period islonger than four weeks. In an embodiment the amount of IgG4 in thepatient's blood is below 200 μg/ml, the determined dose of natalizumabis below 300 mg by IV infusion, and the determined dosage period islonger than four weeks. In an embodiment the amount of IgG4 in thepatient's blood is below 100 μg/ml and the determined dose ofnatalizumab is below 300 mg by IV infusion. In an embodiment the amountof IgG4 in the patient's blood is below 100 ug/ml and the determineddosage period is longer than four weeks. In an embodiment the amount ofIgG4 in the patient's blood is below 100 μg/ml, the determined dose ofnatalizumab is below 300 mg by IV infusion, and the determined dosageperiod is longer than four weeks. In an embodiment the amount of IgG4 inthe patient's blood is below 15 μg/ml and the determined dose ofnatalizumab is below 300 mg by IV infusion. In an embodiment the amountof IgG4 in the patient's blood is below 15 ug/ml and the determineddosage period is longer than four weeks. In an embodiment the amount ofIgG4 in the patient's blood is below 15 μg/ml, the determined dose ofnatalizumab is below 300 mg by IV infusion, and the determined dosageperiod is longer than four weeks. In an embodiment the amount of IgG4 inthe patient's blood is below 200 μg/ml, the determined dose ofnatalizumab is below 300 mg by IV infusion, and the determined dosageperiod is shorter than four weeks. In an embodiment the amount of IgG4in the patient's blood is below 100 μg/ml, the determined dose ofnatalizumab is below 300 mg by IV infusion, and the determined dosageperiod is shorter than four weeks. In an embodiment the amount of IgG4in the patient's blood is below 15 μg/ml, the determined dose ofnatalizumab is below 300 mg by IV infusion, and the determined dosageperiod is shorter than four weeks.

In an embodiment of the method the disease is multiple sclerosis. In anembodiment the multiple sclerosis is selected from relapsing remitting,secondary progressive, primary progressive, and chronic progressivemultiple sclerosis. In an embodiment of the method the disease isinflammatory bowel disease or rheumatoid arthritis. In an embodiment theinflammatory bowel disease is Crohn's Disease.

In an embodiment the method further includes monitoring the patient forindicators of serious infection and/or treating the patient withprophylaxis designed to reduce the risk of developing serious infection.

In an embodiment the method further includes monitoring the patient forindications of progressive multifocal leukoencephalopathy. In anembodiment the monitoring detects JCV in the patient's urine, blood,and/or cerebrospinal fluid. In an embodiment the monitoring comprisesserially removing samples of the patient's blood, measuring the amountof IgG antibodies to JCV in the samples, and comparing the amount of theantibodies in the samples. In an embodiment the monitoring furthercomprises measuring the amount of IgM antibodies to JCV in the samples,and comparing the amount of the IgM and IgG antibodies in the samples.In an embodiment the monitoring detects seroconversion and/or anincreasing titer of JCV in the patient's urine and/or blood, and furtherincludes removing a sample of the patient's cerebrospinal fluid when thecomparison of the serial urine and/or blood samples detectseroconversion and/or an increasing titer of JCV; and testing thecerebrospinal fluid for the presence of JCV. In an embodiment themonitoring includes testing for clinical and/or radiologic symptoms ofprogressive multifocal leukoencephalopathy. In an embodiment the testingfor clinical symptoms comprises testing for new or worseningneurological symptoms. In an embodiment the neurological symptomscomprise one or more of central blindness, mental confusion, personalitychange, and dyskinesia. In an embodiment the testing for radiologicsymptoms comprises performing a Gd-enhanced magnetic resonance imagingscan. In an embodiment the method further includes, in the presence ofindicators of progressive multifocal leukoencephalopathy, providing atleast one treatment selected from intravenous immunoglobulin therapy,plasmapheresis, and antiviral therapy. In an embodiment the antiviraltherapy comprises the administration of at least one therapeuticallyeffective dose of an antiviral agent selected from cytosine arabinoside(cytarabine), cidofovir, and a serotonin antagonist. In an embodimentthe serotonin antagonist is a 5HT2a antagonist.

In an embodiment of the method the patient is not treated simultaneouslywith natalizumab and an immunosuppressive or antineoplastic agent. In anembodiment the immunosuppressive or antineoplastic agent is selectedfrom one or more of chlorambucil, melphalan, 6-mercaptopurine, thiotepa,ifodfamide, dacarbazine, procarbazine, temozolomide, hexamethylmelamine,doxorubicine, daunarubicine, idarubicin, epirubicin, irinotecan,methotrexate, etoposide, vincristine, vinblastine, vinorelbine,cytarabine, busulfan, amonifide, 5-fluorouracil, topotecan, mustargen,bleomycin, lomustine, semustine, mitomycin C, mutamycin, cisplatin,carboplatin, oxaliplatin, methotrexate, trimetrexate, raltitrexid,flurorodeoxyuridine, capecitabine, ftorafur, 5-ethynyluracil,6-thioguanine, cladribine, pentostatin, teniposide, mitoxantrone,losoxantrone, actinomycin D, vindesine, docetaxel, amifostine,interferon alpha, tamoxefen, medroxyprogesterone, megestrol, raloxifene,letrozole, anastrzole, flutamide, bicalutamide, retinoic acids, arsenictrioxide, rituximab, CAMPATH-1, mylotarg, mycophenolic acid, tacrolimus,glucocorticoids, sulfasalazine, glatiramer, fumarate, laquinimod,FTY-720, interferon tau, daclizumab, infliximab, IL10, anti-IL2 receptorantibody, anti-IL-12 antibody, anti-IL6 receptor antibody, CDP-571,adalimumab, entaneracept, leflunomide, anti-interferon gamma antibody,abatacept, fludarabine, cyclophosphamide, azathioprine, cyclosporine,intravenous immunoglobulin, 5-ASA (mesalamine), and a β-interferon.

In another aspect the invention provides a method of using natalizumabto treat a patient with an inflammatory or autoimmune disease bydetermining the amount of IgG4 in the patient's plasma or serum;administering a dose of natalizumab for a first dosage period;monitoring the level of bivalent natalizumab in the patient's plasma orserum during the first dosage period; determining a second dose anddosage period of natalizumab based on the amount of IgG4 in thepatient's plasma or serum and on the level of bivalent natalizumab inthe patient's plasma or serum; and administering the second dose ofnatalizumab for the second dosage period; wherein the second dose anddosage period improve the safety and/or efficacy of the treatment. In anembodiment of the method, the monitoring shows that the amount ofbivalent natalizumab in the patient's plasma or serum remains above apredetermined level during the first dosage period, and the second doseof natalizumab administered over the second dosage period is designed toachieve a reduction of the natalizumab level during the second dosageperiod to below the predetermined level during at least a portion of thesecond dosage period. In an embodiment the first dose of natalizumab is300 mg administered by IV infusion for a first dosage period of fourweeks. In an embodiment the predetermined level is about 1 μg/ml. In anembodiment the predetermined level is about 0.5 μg/ml. In an embodimentthe predetermined level is about 0.1 μg/ml. In an embodiment the amountof IgG4 in the patient's blood is below 200 μg/ml and the determineddose of natalizumab is below 300 mg by IV infusion. In an embodiment thepatient's blood is below 200 ug/ml and the determined dosage period islonger than four weeks. In an embodiment the amount of IgG4 in thepatient's blood is below 200 μg/ml, the determined dose of natalizumabis below 300 mg by IV infusion, and the determined dosage period islonger than four weeks. In an embodiment the amount of IgG4 in thepatient's blood is below 100 μg/ml and the determined dose ofnatalizumab is below 300 mg by IV infusion. In an embodiment the amountof IgG4 in the patient's blood is below 100 ug/ml and the determineddosage period is longer than four weeks. In an embodiment the amount ofIgG4 in the patient's blood is below 100 μg/ml, the determined dose ofnatalizumab is below 300 mg by IV infusion, and the determined dosageperiod is longer than four weeks. In an embodiment the amount of IgG4 inthe patient's blood is below 15 μg/ml and the determined dose ofnatalizumab is below 300 mg by IV infusion. In an embodiment the amountof IgG4 in the patient's blood is below 15 ug/ml and the determineddosage period is longer than four weeks. In an embodiment the amount ofIgG4 in the patient's blood is below 15 μg/ml, the determined dose ofnatalizumab is below 300 mg by IV infusion, and the determined dosageperiod is longer than four weeks. In an embodiment the standard dose ofnatalizumab is 300 mg IV infusion and the standard dosage period is fourweeks.

In another embodiment of the method the corrected dose is lower than thefirst dose, or the second dosage period is longer than the first dosageperiod, or the corrected dose is lower than the first dose and thesecond dosage period is longer than the first dosage period. In anembodiment the amount of bivalent natalizumab in the patient's plasma orserum falls below a predetermined level during the first dosage periodwithin a predetermined time after administration of the first dose, andwherein the second dose of natalizumab administered over the seconddosage period is designed to maintain the natalizumab level above thepredetermined level at least up to the predetermined time afteradministration of the second dose during the second dosage period.

In another embodiment the method further includes monitoring the patientfor indicators of serious infection. In an embodiment the seriousinfection is progressive multifocal leukoencephalopathy. In anembodiment the method further includes treating the patient withprophylaxis designed to reduce the risk of developing serious infection.In an embodiment the serious infection is progressive multifocalleukoencephalopathy.

In another aspect the invention provides a method of using natalizumabto treat a patient with an inflammatory or autoimmune disease bydetermining the amount of IgG4 in the patient's plasma or serum prior toinitiating treatment; initiating treatment of the patient withnatalizumab in the event the amount of IgG4 in the patients plasma orserum is above a predefined threshold; and initiating treatment of thepatient with natalizumab with increased monitoring for indicators ofprogressive multifocal leukoencephalopathy and/or opportunisticinfections in the event the amount of IgG4 in the patients plasma orserum is at or below a predefined threshold; wherein determining of theamount of IgG4 in the patient's plasma or serum improves the safetyand/or efficacy of the treatment. In an embodiment of the method theamount of IgG4 in the patients plasma or serum is at or below a secondpredefined threshold, treatment is not initiated. In an embodimenttreatment is initiated if the amount of IgG4 in the patient's blood isabout 200 μg/ml or higher. In an embodiment treatment is initiated ifthe amount of IgG4 in the patient's blood is about 100 μg/ml or higher.In an embodiment treatment is initiated if the amount of IgG4 in thepatient's blood is about 15 μg/ml or higher.

In an embodiment the method further includes determining the amount ofIgG4 in the patient's plasma or serum during treatment; and terminatingtreatment in the event the amount of IgG4 is the patients plasma orserum is below a predefined threshold. In an embodiment treatment isterminated if the amount of IgG4 in the patient's plasma or serum isabout 200 μg/ml or lower. In an embodiment treatment is terminated ifthe amount of IgG4 in the patient's plasma or serum is about 100 μg/mlor lower. In an embodiment treatment is terminated if the amount of IgG4in the patient's plasma or serum is about 15 μg/ml or lower.

In an embodiment the method further includes monitoring the amount ofbivalent natalizumab in the patient's plasma or serum during treatment;and terminating treatment in the event the amount of bivalentnatalizumab is above a predefined threshold. In an embodiment treatmentis terminated if the amount of bivalent natalizumab in the patient'splasma or serum is about 1 μg/ml or higher. In an embodiment treatmentis terminated if the amount of bivalent natalizumab in the patient'splasma or serum is about 0.5 μg/ml or higher. In an embodiment treatmentis terminated if the amount of bivalent natalizumab in the patient'splasma or serum is about 0.1 μg/ml or higher.

In an embodiment the method further includes monitoring the amount ofbivalent natalizumab in the patient's plasma or serum during treatment;and terminating treatment in the event the amount of bivalentnatalizumab is above a predefined threshold. In an embodiment treatmentis terminated if the amount of bivalent natalizumab in the patient'splasma or serum is about 1 μg/ml or higher. In an embodiment treatmentis terminated if the amount of bivalent natalizumab in the patient'splasma or serum is about 0.5 μg/ml or higher. In an embodiment treatmentis terminated if the amount of bivalent natalizumab in the patient'splasma or serum is about 0.1 μg/ml or higher. In an embodimentintravenous immunoglobulin is administered to the patient to reduce thelevel of bivalent natalizumab in the patient's plasma or serum. In anembodiment plasmapheresis therapy is administered to the patient toreduce the level of bivalent natalizumab in the patient's plasma orserum.

In another aspect the invention provides a method of treating a patientwith an inflammatory or autoimmune disease with natalizumab byadministering a dose of natalizumab for a first dosage period;monitoring the amount of bivalent natalizumab in the patient's plasma orserum during the first dosage period; determining a second dose ofnatalizumab based on the level of bivalent natalizumab observed;administering a second dose of natalizumab for a second dosage period;and administering the second dose of natalizumab for one or moresubsequent second dosage periods; wherein the second dose improves thesafety and/or efficacy of the treatment during the second dosage period.

In another aspect the invention provides a method of treating a patientwith an inflammatory or autoimmune disease with natalizumab bydetermining the amount of IgG4 in the patient's plasma or serum;determining a dose and dosage period of natalizumab based on the amountof IgG4 in the patient's plasma or serum; administering the dose ofnatalizumab to the patient over the dosage period; and administering thedose of natalizumab to the patient over one or more subsequent dosageperiods; wherein the dose and dosage period improve the safety and/orefficacy of the treatment compared to the safety and/or efficacyprovided by the standard dose and dosage period of natalizumab.

In another aspect the invention provides a method of using natalizumabto treat a patient with an inflammatory or autoimmune disease bydetermining the amount of IgG4 in the patient's plasma or serum;administering a dose of natalizumab for a first dosage period;monitoring the level of bivalent natalizumab in the patient's plasma orserum during the first dosage period; determining a second dose anddosage period of natalizumab based on the amount of IgG4 in thepatient's plasma or serum and on the level of bivalent natalizumab inthe patient's plasma or serum; administering the second dose ofnatalizumab for the second dosage period; and administering the seconddose of natalizumab for one or more subsequent second dosage periods;wherein the second dose and dosage period improve the safety and/orefficacy of the treatment.

DESCRIPTION OF THE INVENTION Definitions

The terms used herein have their ordinary meanings, as set forth below,and can be further understood in the context of the specification.

A “patient” or “subject,” used interchangeably herein, is a human unlessotherwise indicated.

“Treatment” means any administration or application of remedies fordisease and includes inhibiting the disease, arresting its development,and relieving the disease, for example, by causing regression, orrestoring or repairing a lost, missing, or defective function or bystimulating an inefficient process.

“Terminating” means either a temporary or a permanent cessation.

“Dose” means the amount of natalizumab administered to a patient.

“Dosage period” means the time between the administration of a dose andthe next successive administration of a dose. The dosage period maychange with one or more further successive dose or doses, or may remainconstant.

“Natalizumab” or “Natalizumab®” is a humanized antibody against VLA-4 asdescribed in U.S. Pat. Nos. 5,840,299 and 6,033,665, which are hereinincorporated by reference in their entireties. Also contemplated hereinare other antibodies specific for VLA-4, including, but not limited to,immunoglobulins described in U.S. Pat. Nos. 6,602,503 and 6,551,593, andpublished U. S. Application No. 20020197233 by Relton et al. Theseantibody can be prepared by the methods disclosed in these documents, bymammalian cell expression systems, and by transgenic animal expressionsystems, for example, transgenic goats.

A “pharmaceutically effective amount” or “therapeutically effectiveamount,” used interchangeably, is an amount sufficient to cure or atleast partially arrest the symptoms of a disease and/or thecomplications of a disease.

A “serotonin antagonist” is any substance that decreases one or moreeffect of serotonin.

“Seroconversion” is the change of a serologic test from negative topositive, indicating the development of antibodies.

“Titer” is the concentration of an antibody in solution.

IgG4 Antibodies

Antibodies are proteins used by the immune system to identify andneutralize foreign objects like bacteria and viruses. Each antibodyrecognizes a specific antigen unique to its target. Immunoglobulins areglycoproteins in the immunoglobulin superfamily that function asantibodies. They are synthesized and secreted by plasma cells that arederived from the B cells of the immune system. B cells are activatedupon binding to their specific antigen and differentiate into plasmacells. In some cases, the interaction of the B cell with a T helper cellis also necessary.

Immunoglobulins are heavy plasma proteins, often with added sugar chainson N-terminal (all antibodies) and occasionally O-terminal (IgA1 andIgD) amino acid residues. The basic unit of each antibody is a monomer.An antibody can be monomeric, dimeric, trimeric, tetrameric, pentameric,etc. The monomer is a “Y”-shape molecule that consists of two identicalheavy chains and two identical light chains connected by disulfidebonds.

There are five types of heavy chain: γ, δ, α, ρ, and ε. They defineclasses of immunoglobulins. Heavy chains α and γ have approximately 450amino acids, while μ and c have approximately 550 amino acids. Eachheavy chain has a constant region, which is the same by allimmunoglobulins of the same class, and a variable region, which differsbetween immunoglobulins of different B cells, but is the same for allimmunoglobulins produced by the same B cell. Heavy chains γ, α and δhave the constant region composed of three domains but have a hingeregion; the constant region of heavy chains μ and ε is composed of fourdomains. The variable domain of any heavy chain is composed of onedomain. These domains are about 110 amino acids long. There are alsosome amino acids between constant domains. There are only two types oflight chain: λ, and κ. In humans, they are similar, but only one type ispresent in each antibody. Each light chain has two successive domains:one constant and one variable domain. The approximate length of a lightchain is from 211 to 217 amino acids.

The monomer is composed of two heavy and two light chains. Together thisgives six to eight constant domains and four variable domains. Enzymaticcleavage with papain creates two Fab (fragment antigen binding)fragments and an Fc (fragment crystallizable) fragment, whereas pepsincleaves below hinge region, so a f(ab)2 fragment and a fc fragment isformed. Thus, each half of the forked end of the “Y”-shape monomer iscalled the Fab fragment. It is composed of one constant and one variabledomain of each the heavy and the light chain, which together shape theantigen binding site at the amino terminal end of the monomer. The twovariable domains bind the antigens they are specific for and thatelicited their production.

The Fc fragment is composed of two heavy chains that each contribute twoto three constant domains (depending on the class of the antibody). Itbinds to various cell receptors and complement proteins. In this way, itmediates different physiological effects of antibodies (opsonization,cell lysis, mast cell, basophil and eosinophil degranulation and otherprocesses). The variable regions of the heavy and light chains can befused together to form a single chain variable fragment (scFv), whichretains the original specificity of the parent immunoglobulin.

Immunoglobulins are grouped into five classes, or isotypes, based ondifferences in heavy chain constant domains: IgG, IgA, IgM, IgD, andIgE. (The isotypes are also defined with light chains.) Other immunecells partner with antibodies to eliminate pathogens depending on whichIgG, IgA, IgM, IgD, and IgE constant binding domain receptors it canexpress on its surface.

The antibodies that a single B lymphocyte produces can differ in theirheavy chain and the B cell often expresses different classes ofantibodies at the same time. However, they are identical in theirspecificity for antigen, conferred by their variable region. To achievethe large number of specificities the body needs to protect itselfagainst many different foreign antigens, it must produce millions of Blymphocytes.

IgG is a monomeric immunoglobulin, built of two heavy chains γ and twolight chains. Each molecule has two antigen binding sites. This is themost abundant immunoglobulin and is approximately equally distributed inblood and in tissue liquids. This is the only isotype that can passthrough the placenta, thereby providing protection to the fetus in itsfirst weeks of life before its own immune system has developed. It canbind to many kinds of pathogens, for example viruses, bacteria, andfungi, and protects the body against them by complement activation(classic pathway), opsonization for phagocytosis and neutralisation oftheir toxins. There are 4 subclasses: IgG1 (66%), IgG2 (23%), IgG3 (7%)and IgG4 (4%). IgG1, IgG3 and IgG4 cross the placenta easily. IgG3 isthe most effective complement activator, followed by IgG1 and then IgG2.IgG4 does not activate complement. IgG1 and IgG3 bind with high affinityto Fc receptors on phagocytic cells. IgG4 has intermediate affinity andIgG2 affinity is extremely low.

Immunoglobulin G4 (IgG4) antibodies have been known for some time to befunctionally monovalent. Recently, the structural basis for thismonovalency has been elucidated: the in vivo exchange of IgGhalf-molecules (one H-plus one L-chain) among IgG4. This process resultsin bispecific antibodies that in most situations will behave asfunctionally monovalent antibodies. The structural basis for theabnormal behaviour of IgG4 seems to be largely the result of a singleamino acid change relative to human IgG1: the change of a proline incore hinge of IgG1 to serine. This results in a marked shift in theequilibrium between interchain disulphide bridges and intrachaindisulphide bridges, which for IgG4 results in 25-75% absence of acovalent interaction between the H-chains. Because of strongnon-covalent interactions between the CH3 domains (and possibly alsobetween the CH1 domain and the trans-CH2 domain) IgG4 is a stablefour-chain molecule and does not easily exchange half-molecules understandard physiological conditions in vitro. The exchange may becatalysed in vivo by protein disulphide isomerase (PDI) and/or FcRn (themajor histo-compatibility complex (MHC)-related Fc receptor) duringtransit of IgG4 in the endosomal pathway in endothelial cells, or by anunknown mechanism. Because IgG4 is predominantly expressed underconditions of chronic antigen exposure, the biological relevance of thisexchange of half-molecules is that it generates antibodies that areunable to form large immune complexes and therefore have a low potentialfor inducing immune inflammation. In contrast to monovalentimmunoglobulin fragments, these scrambled immunoglobulins have a normalhalf-life. The significance of the ensuing bispecificity needs furtherevaluation, because this will be relevant only in situations where highIgG4 responses are found to two unrelated antigens that happen to bepresent in the body at the same time and place. In this context thesignificance of IgG4 autoreactivity might have to be re-evaluated. Themain function of IgG4, however, is presumably to interfere with immuneinflammation induced by complement-fixing antibodies, or, in the case ofhelminth infection or allergy, by IgE antibodies.

Most monoclonal antibodies consist of one type of L- and H-chain andhave two identical antigen binding sites, which makes each monoclonalantibody molecule bivalent. In the case of IgG4, however, half-moleculeexchange in vivo creates antibodies that are bispecifically monovalent.The experimental evidence supporting this view of IgG4 structure in vivoincludes the observations that polyclonal IgG4 antibodies do notcrosslink two antigens, i.e. are functionally monovalent; in contrast topolyclonal IgG4 antibody, monoclonal (chimeric) IgG4 antibody doescrosslink two antigens; a substantial fraction of IgG4 (both monoclonaland polyclonal) lacks a covalent interaction between the heavy chains,but is maintained as a four-chain structure solely via non-covalentbonds; and bispecific antibodies can be found in plasma, which aremostly, if not exclusively, of the IgG4 type. Quantitatively, the levelof bispecific reactivity can be predicted from the level ofantigen-specific IgG4 antibodies.

Natalizumab

Natalizumab is a recombinant humanized IgG₄κ monoclonal antibodydirected against the α4-integrins α4β1 and α4β7. Natalizumab containshuman framework regions and the complementarity-determining regions of amurine antibody that binds to a4-integrin. The molecular weight ofnatalizumab is 149 kilodaltons.

Studies by Yednock and others have shown the clinical efficacy ofα4-integrin blockade in experimental allergic encephalomyelitis (EAE),an animal model of MS (Yednock et al., Nature 1992; 356:63-66 (1992);Baron et al., J. Exp. Med. 177:57-68 (1993); Kent et al., J.Neuroimmunol. 58:1-10 (1995); Brocke et al., Proc. Natl. Acad. Sci.96:6896-6901 (1999). These data demonstrated that α4-integrin blockadeby a bound antibody can prevent leukocyte migration into the brain andthus support the hypothesis that α4-integrins are a target for MStherapeutics. In addition, these observations support the hypothesisthat blockading leukocyte accumulation in the brain will prevent thelocal destruction of myelin, the insulating sheath covering nervefibers, and neurons, which characterizes MS lesions. Natalizumab is thefirst antibody directed at this target and clinical data demonstrate therelevance of this treatment strategy.

Natalizumab is a member of an emerging class of agents known as theSelective Adhesion Molecule (SAM) Inhibitors. Natalizumab binding toα4β1 (also called VLA-4) and α4β7 integrins inhibits their molecularinteractions with cognate integrin receptors on endothelial cells,VCAM-1 and MAdCAM-1, respectively. By inhibiting these molecularinteractions, natalizumab prevents the recruitment and egress ofleukocytes into sites of inflammation. A further mechanism ofnatalizumab action may be to suppress ongoing inflammatory reactions indiseased tissues by inhibiting the interaction of α4-expressingleukocytes with other ligands in the extracellular matrix (osteopontinand fibronectin) and on parenchymal cells, such as microglial cells(VCAM-1). As such, natalizumab may suppress ongoing inflammatoryactivity at the disease site and inhibit further recruitment of immunecells into inflamed tissues. Thus, treating MS patients with natalizumabmay block entry of mononuclear leukocytes into the CNS and attenuate theinflammatory process that results in demyelination and axonal damage andultimately provide clinical benefit by reducing the number of clinicalrelapses and the progression of disability, including motor, visual, andcognitive function.

Pharmacokinetics of Natalizumab

Following the repeat intravenous administration of a 300 mg dose ofnatalizumab to multiple sclerosis patients, the mean maximum observedserum concentration was 98±34 μg/mL. Mean average steady-statenatalizumab concentrations over the dosing period were approximately 30μg/mL. The mean half-life of 11±4 days was observed with a clearance of16±5 mL/hour. The distribution volume of 5.7±1.9 L was consistent withplasma volume.

As measured in an in vitro receptor saturation assay, natalizumab,straight from the vial, saturates lymphocytes in whole blood at 0.3 to 1ug/ml. This result is consistent with observations on cell adhesion,where stringent adhesion requires 1 ug/ml natalizumab. However, usingthe same assay to examine natalizumab in patient serum samples, receptorsaturation appeared to require greater than 10 ug/ml. This effect mayresult from natalizumab swapping one of its IgG4 arms with other IgG4antibodies in the serum in vivo, resulting in a loss of avidity andpotency. Based on work with other single chain antibodies, this changecould easily affect potency by 100-fold. Therefore, any meaningfulnatalizumab activity in the blood would most likely come from bivalentmolecules remaining in the circulation after equilibrium was establishedwith endogenous IgG4. Data indicates that this process appears to occurin a stoichiometric fashion over a relatively short period of time(hours to days). Thus, within hours or days the level of bivalentnatalizumab present depends upon the starting level of endogenous IgG4.

A typical range for endogenous IgG4 is about 200 to 1000 ug/ml inhumans. Based on calculations of the stoichiometry, when natalizumab ispresent at 10 ug/ml (the typical nadir blood level following a 300 mgdose administered IV over a four week dosage period), the level ofbivalent natalizumab would range from 0.02 to 0.24 ug/ml at nadir levels(averaging 0.12 ug/ml). These values fit very well with the 75-85%receptor saturation levels observed with natalizumab in patient samplesat nadir levels. Functional saturation with bivalent natalizumab occursat 1 ug/ml. The level of bivalent natalizumab, however, increasessignificantly with lower levels of endogenous IgG4. For example,assuming a nadir level of natalizumab at 10 ug/ml, in a patient with 50ug/ml of endogenous IgG4 the level of bivalent natalizumab would be ˜1ug/ml (or saturation). So patients with levels of endogenous IgG4 lowerthan about 50 ug/ml, for example, may have chronically saturating levelsof natalizumab throughout the dosing period. At an IgG4 concentration ofabout 15 ug/ml the level of fully functional, bivalent natalizumab atnadir levels would be 2.5 ug/ml, well above receptor saturation. If anindividual lacked IgG4 entirely, functional levels of natalizumab wouldbe the same as the measured nadir levels, or about 10 ug/ml in a patientreceiving a 300 mg dose by IV infusion over a four week dosing period.

As the exemplary calculations above make clear, a patient with a levelof endogenous IgG4 equal to or below a certain level, such as about 200ug/ml, about 100 ug/ml, about 50 ug/ml, about 15 ug/ml, or about zerowould likely have a very different clinical profile with natalizumabthan patients with normal levels of endogenous IgG4 (more that about 1ug/ml functional antibody vs. 0.12 ug/ml).

These calculations also demonstrate that natalizumab exhibits strongefficacy even though concentrations drop below full occupancy levels forone or two weeks every month in typical patients, receiving a standarddose of 300 mg by IV infusion over a four week dosing period. Thisobservation suggests that cell traffic into the CNS of typical patientsis only partially inhibited during part of each dosing period. However,in a minority of patients, particularly those with low levels ofendogenous IgG4, higher nadir levels of bivalent natalizumab may resultin prolonged receptor saturation with natalizumab, resulting in completeinhibition of α4-integrins α4β1 and α4β7 for the dosing period and moreprofound inhibition cell trafficking. One consequence of this conditionis that those patients are at a higher risk of serious infection whilebeing treated with natalizumab.

Any factor that alters the nadir level of total natalizumab during adosage interval will also affect the nadir level of bivalent, fullyactive natalizumab antibody. For example, in a patient with 100 ug/ml ofendogenous IgG4, if nadir natalizumab levels are doubled, from 10 ug/mlto 20 ug/ml, then the level of bivalent antibody would triple,increasing from 0.5 ug/ml (below saturation) to 1.8 ug/ml (abovesaturation). Factors in addition to IgG4 levels that could affect thelevel of bivalent, fully active natalizumab antibody present during adosing period include body weight, concomitant medication, and treatmentduration. When using a fixed dose of natalizumab, differences in bodyweight can affect natalizumab levels by up to three-fold, e.g., 3 vs. 9ug/ml or 6 vs. 16 ug/ml.

Although the effect of AVONEX® on pharmacokinetics is disputed, dataderived from a small subset of patients indicate that nadir levels ofnatalizumab were 12 ug/ml with natalizumab alone (which is in line withall other studies) vs. 25 ug/ml when natalizumab is coadministered withAVONEX®. This is a difference that again, could triple the level ofbivalent natalizumab molecules. Even if this effect isn't statisticallysignificant for the population at large—if AVONEX® affects the upperlimit of the range or nadir concentrations of natalizumab for even asmall number of individuals, it could cause a significant change in therisk profile across the total population of patients.

Natalizumab may be administered repeatedly, such as at four weekintervals. The number of prior doses of natalizumab may affect nadirlevels experienced during each subsequent dosage interval. For example,concentration levels appear to increase with repeat dosing 5 ug/mlbefore dose number two vs. 12 ug/ml for dose number 15. Thus, Tysabrimay accumulate with repeated dosing.

Methods of Treatment

Pharmaceutical compositions of natalizumab will be administeredintravenously. The dose of natalizumab administered and the dosageperiod may be fixed across all or a class of patients or may bedetermined based on patient weight. For example, in an embodimentnatalizumab is administered at a dose of from one to five mg per kg bodyweight by IV infusion. Alternatively, a fixed dose of natalizumab may beadministered to all patients or to a class of patients independent ofthe body weight of the patients. For example, in an embodimentnatalizumab is administered at a dose of 300 mg by IV infusion.

In an embodiment, the dose, either weight-based or fixed, is determinedor adjusted based on the amount of bivalent natalizumab in the patient'splasma or serum and/or the amount of IgG4 in the patient's plasma orserum.

In an embodiment the amount of bivalent natalizumab in the patient'splasma or serum and/or the amount of IgG4 in the patient's plasma orserum are determined during a dosing period. The amount of bivalentnatalizumab may be determined directly or may be determined or estimatedindirectly, such as by measuring the amount of IgG4 and/or totalnatalizumab in the patient's plasma or serum and calculating orestimating the amount of bivalent natalizumab on the basis of thosemeasurements.

In an embodiment the amount of IgG4 in the patient's plasma or serum maybe used to determine a suitable dose and dosage period foradministration of natalizumab, either prior to and/or after initiationof treatment. If the amount of IgG4 in the patient's blood is below 200μg/ml, below 100 ug/ml, below 15 ug/ml, or lower, such as undetectable,the dose of natalizumab determined on the basis of the amount of IgG4 inthe patient's plasma or serum may be lower than the standard dose orlower than the dose previously given to the patient. Also, thedetermines dosage period may be longer than the standard dosage period,or longer than one ore more previously scheduled dosage periods. Forexample, the determined dose may be below 300 mg by IV infusion, thedetermined dosage period may be longer than four weeks, or thedetermined dose may be below 300 mg by IV infusion and the determineddosage period may be longer than four weeks.

In an embodiment the amount of bivalent natalizumab in a patient'splasma or serum during a first dosage period may be used to determine asecond dose of natalizumab for administration for a second dosageperiod. For example, if the monitoring shows that the amount of bivalentnatalizumab in the patient's plasma or serum remains above apredetermined level during the first dosage period, the corrected doseof natalizumab administered over the second dosage period can bedesigned to achieve a reduction of the natalizumab level during thesecond dosage period to below the predetermined level during at least aportion of the second dosage period. This can be achieved, for example,by determining a second dose lower than the first dose, a second dosageperiod longer than the first dosage period, or by determining a seconddose lower than the first dose and a second dosage period longer thanthe first dosage period. For example, the predetermined level may beabout 1 ug/ml, about 0.5 ug/ml, or about 0.1 ug/ml. The second dose ofnatalizumab may be below a standard dose and/or the determined dosageperiod may be longer than a standard dosage period. For example, thedetermined dose may be below 300 mg by IV infusion, the determineddosage period may be longer than four weeks, or the determined dose maybe below 300 mg by N infusion and the determined dosage period may belonger than four weeks.

ELISA Assays for Total and Bivalent Natalizumab

The amount of total natalizumab in a solution, for example a biologicalfluid such as plasma or serum, can be measured using an Enzyme-LinkedImmunosorbant Assay (ELISA). The invention provides ELISA assays thatmeasure both monovalent and bivalent natalizumab. Specifically, itprovides solid-phase sandwich ELISAs for measuring the concentration oftotal natalizumab and the concentration of bivalent natalizumab.Generally, the ELISA assays described herein utilize antiidiotypeantibodies specific for natalizumab and enzyme-linked antibodies to theFc region of IgG4 antibodies.

In both the total natalizumab and bivalent natalizumab ELISAs, theantiidiotype antibody is bound to a solid surface. Any antiidiotypeantibody specific for natalizumab is suitable for use in the ELISA. Theantibody 12C4 is an example of an antiidiotype antibody specific for thenatalizumab variable region and suitable for use in the assay. Suitablesolid surfaces are well known in the art and include microtiter plates.

In the ELISA assay for total natalizumab, the antiidiotype antibody isbound to the surface at high density. In an embodiment, 12C4 is bound ata density of approximately 2 ug/ml. A fluid solution comprising anunknown quantity of natalizumab is added and allowed to interact withthe antiidiotype antibody bound to the surface under conditionssufficient for natalizumab in the solution to bind to the antiidiotypeantibody. The unbound portion of the solution is removed by washing thesurface. The natalizumab which bound to the antiidiotype antibody isthen detected using an antibody which is specific for the Fc region ofIgG4 antibodies and is conjugated, directly or indirectly, to an enzyme.The surface is again washed to remove unbound enzyme-conjugated IgG4. Asubstrate for the enzyme is added and the reaction product is measured.The amount of reaction product correlates with the amount of natalizumabin solution. Suitable enzymes, substrates, and measuring devices arewell-known in the art. In an embodiment, a commercially availableantibody to IgG4 conjugated to alkaline phosphatase is used to detectbound natalizumab.

The amount of bivalent natalizumab can also be measured by solid phasesandwich ELISA using methods provided herein. An antiidiotype antibodyis bound to a solid surface at low density. In an embodiment, 12C4 isbound to the surface at a density of approximately 0.3 ug/ml. A solutioncomprising an unknown quantity of natalizumab is allowed to interact asdescribed above for the total natalizumab ELISA. The natalizumab is thendetected with an antiidiotype antibody specific for bivalentnatalizumab, which does not recognize either natalizumab monomers insolution or natalizumab monomers which has exchanged an IgG4 heavy chainwith endogenous IgG4 and remain bound to the endogenous IgG4. Theantiidiotype antibody used in the detection step is conjugated to adetectable enzyme. The antiidiotype antibody 12C4 is suitable for use inthe bivalent natalizumab assay both as the solid phase bound antibodyand/or as the detecting antibody. When used as the detecting antibody,12C4 is conjugated to a detectable enzyme.

A wide variety of enzyme detection systems are known in the art. Theyinclude alkaline phosphatase conjugates, avidin and streptavidinconjugates, horseradish peroxidase conjugates, beta-galactosidaseconjugates, and the like. A wide variety of substrates are also wellknown in the art and include 4-nitrophenylphosphate,2-nitrophenyl-b-D-galactopyranoside,2,2′-azino-di-[3-ethylbenzthiazoline sulfonate].

The concentration of both total and bivalent natalizumab in the serum ofmonkeys treated with natalizumab was determined using the ELISA assaysof the invention. Natalizumab was measured 4, 24, and 72 hours afterdosing with 3 mg/kg. The results of the 4 hr and 24 hr measurements areshown in Table 1. The acronym “blq” is used to denote that the resultwas below the limit of quantification. Serum spiked with 10 ug/mlnatalizumab was used as a positive control.

TABLE 1 Total and Bivalent Serum Natalizumab Levels Experiment 1Experiment 2 Experiment 3 4 hr 24 hr Total Bivalent Total BivalentBivalent Animal Nat. Nat. Nat. Nat. Nat. 1 56.1 37.3 17.4 48.3 16.7 253.0 32.6 14.1  1.0 0.5 3 21.1 47.0 16.9 4 66.8 31.0 10.8  0.3 1.8 565.4 38.0 19.8 41.7 15.2 6 8.9  0.5 0.3 7 54.6 30.0 7.3 blq 0.2 8 53.027.8 9.6 blq 0.2 9 17.3 14.2 5.4 10 54.1 37.4 12.9 29.0 6.1 11 57.4 28.510.1 0.4-3 0.2 12 18.7 35.2 18.7

Safety of Natalizumab

The safety of natalizumab is demonstrated herein, based on results oftreating 3,919 subjects with natalizumab in clinical trials for MS,Crohn's Disease, and rheumatoid arthritis, resulting in 5,505patient-years of natalizumab exposure. Treatment with natalizumab wasgenerally well tolerated. Eighteen treatment-emergent deaths occurred inthe entire natalizumab program. The adverse events encountered in thetrials, both common and serious, were similar in natalizumab-treatedpatients and controls. Adverse events that led to discontinuation ofnatalizumab occurred in 5.8% of natalizumab-treated MS patients and in4.8% of placebo-treated MS patients, with urticaria being the mostcommon cause of discontinuation in the natalizumab-treated patients(1.2%).

Like other highly active drugs used to treat autoimmune diseases,natalizumab is not without risk. Unfortunately, with the clinicalefficacy of immunomodulatory agents such as natalizumab comes the riskof significant mechanism-based side effects. The risks of medicationsthat modulate immune function in order to treat serious chronic diseaseshave been well recognized over the past several years. Medicines such asthe TNFα antagonists (e.g., infliximab, adalimumab, and etanercept) arepotent modulators of immune function and are approved for numerousserious autoimmune diseases such as rheumatoid arthritis, Crohn'sDisease, psoriasis, psoriatic arthritis, and ankylosing spondylitis.Although very effective, these agents are associated with seriousadverse events, particularly infections that have been associated withsignificant morbidity and mortality.

The invention provides the identification, through detailed safetyanalyses, of PML as a rare, but significant, risk of natalizumabtreatment. In addition, serious non-PML opportunistic infections havebeen observed in natalizumab-treated patients, mostly in Crohn's Diseasepatients in association with concurrent immunosuppressant use or othersignificant co-morbidities. In addition, we have identified patientpopulations in whom the benefit-risk profile is less well defined. Theoccurrence of these infections highlights the need for a comprehensiverisk management program in the post-marketing setting focused onappropriate use conditions and assessment and minimization of the riskof PML and other serious opportunistic infections.

Deaths

Of the eighteen deaths that occurred during the clinical trials, fiveoccurred in the placebo-controlled MS trials, including two in patientswho had received natalizumab and three who had received placebo). Thepatients who received natalizumab died of alcohol intoxication andmetastatic malignant melanoma. The patients who received placebo died ofcardiac arrest, respiratory arrest, and pleural carcinomatosis withseizures. Four deaths occurred in the open-label MS trials, due torespiratory distress, PML, suicide, and seizure due to MS.

Six deaths of natalizumab-treated Crohn's Disease patients were observedin the trials. The exposure to natalizumab was approximately three-foldgreater in these trials than exposure to placebo. The causes of deathwere acute myocardial infarction, acute renal failure, carbon dioxideasphyxiation, PML, Pneumocystis carinii pneumonia, and bronchopulmonaryaspergillosis.

Three deaths occurred in the rheumatoid arthritis trials, two innatalizumab-treated patients and one in a patient treated with aplacebo. The natalizumab-treated patients died of hemoptysis withrespiratory failure and end-stage rheumatoid pulmonary disease. Theplacebo-treated patient died of circulatory and respiratoryinsufficiency.

In the MS studies, apart from PML, no other safety signal was apparentfrom the study deaths. In the Crohn's Disease studies, one patient diedfrom PML. Two additional deaths in Crohn's Disease were associated withopportunistic infections, namely, bronchopulmonary aspergillosis andPneumocystis carinii pneumonia. These patients had significantco-morbidities, which may have contributed to the development of theseinfections.

Adverse Events

At least one serious adverse event was encountered by 251 of the 1,617natalizumab-treated MS patients (15.5%) and by 214 of the 1,135placebo-treated patients (18.9%) in the placebo-controlled trial. Themost common serious adverse events, classified by organ systems, werenervous system disorders (5.9% natalizumab, 10.2% placebo). MS relapsecontributed significantly to this incidence (4.7% natalizumab, 9.0%placebo). The second most common serious adverse events were infectionsand infestations (2.4% natalizumab, 2.2% placebo), with appendicitis andurinary tract infection (<1% in both groups) as the most common.

The incidence of hypersensitivity reactions, an event expected to resultfrom treatment with therapeutic proteins, was approximately 4% withserious systemic reactions occurring at an incidence of less than 1%.The reactions tended to occur early in the treatment course, but wereobserved throughout the course of infusion. Although the specificmechanisms of the reactions have not been determined, clinically, thereactions appeared to be typical IgE- or IgG-mediated immediate-typehypersensitivity reactions. All patients recovered without sequelae.

The occurrence of malignancy during natalizumab treatment was uncommon.The incidence of malignancy was balanced between the natalizumab andcontrol groups. The rates of malignancies observed during natalizumabtreatment were within the expected rates per comparison with theexisting cancer registries, such as the National Cancer Institute'sSurveillance Epidemiology and End Results.

Evaluation of PML Cases

Three confirmed cases of PML have been identified, two of which werefatal. Two cases occurred in MS patients and one in a patient withCrohn's Disease. Both MS patients received natalizumab for over twoyears in addition to AVONEX®. The Crohn's Disease patient received eightdoses of natalizumab over an 18-month period and was immunocompromiseddue to chronic azathioprine use as manifested by persistent lymphopenia.All three PML patients presented with subtle clinical changes early intheir disease course that were noted by the patients or their families.

The first patient to contract a fatal case of PML was a 46-year-oldfemale with MS who presented to her neurologist with right-sidedparesthesia and dysesthesia, and right upper extremity clumsiness. MRIbrain scanning demonstrated four non-enhancing T2-hyperintense lesionsbilaterally in the corona radiata. Six weeks later, she presented withnew blurring of the vision in her right eye. Visual acuity was 20/15 inthe left eye and 20/100 in the right. Spinal fluid analysis yielded onewhite blood cell, normal protein and glucose, and no oligoclonal bands.A follow-up MRI brain scan revealed two new subcortical lesions in theright parietal region that were hyperintense on FLAIR imaging andhypointense on T1.

AVONEX® treatment was initiated, but she subsequently suffered threerelapses, the most recent of which involved band-like pain around theabdomen, lower extremity weakness, and spasticity requiring treatmentwith methylprednisolone. Her Expanded Disability Status Scale (EDSS)score in prior to entry into the placebo-controlled MS study, asdescribed in more detail below, was 2.5. She received 30 infusions ofnatalizumab before entering the open-label extension study and receivingan additional seven infusions. She had no exacerbations or suspectedrelapses during her time in the placebo-controlled study. She developedfive new or enlarging T2-hyperintense lesions during the first year ofthe placebo-controlled study and one during the second year. She wasnegative for anti-natalizumab antibodies and her serum concentration ofnatalizumab was similar to the mean of the study populations throughouther participation.

In November 2004, she began to experience motor dysfunction, andcognitive and language difficulties, which progressed to righthemiparesis by the following month. An MRI brain scan performed inDecember 2004 revealed left frontal T2-hyperintensity andT1-hypointensity with extension into the centrum semiovale and coronaradiata without Gd-enhancement. She received two courses of high dosesteroids over the next few months, but continued to decline. Shereceived her last dose of natalizumab on Jan. 18, 2005. She wasreadmitted to the hospital on Feb. 12, 2005 with worsening clinicalstatus. A repeat MRI brain scan in February 2005 showed extension of thelesion seen previously. An extensive work-up over the next week revealedJC viral DNA in the CSF, resulting in the diagnosis of PML. She died onFeb. 24, 2005. Post-mortem examination revealed normal organs withoutevidence of opportunistic infection. The brain examination revealedextensive, severe cavitation mainly in the left hemisphere as well asmultiple non-cavitated, ovoid areas throughout the white matter of bothhemispheres typical of PML, having reactive astrocytes with enlarged,hyperchromatic nuclei (Kleinschmidt-DeMasters and Tyler, N. Engl. J.Med. 353:369-374 (2005)).

The second patient is a 46-year-old male who experienced his firstsymptoms of relapsing/remitting MS in 1983. His past medical history issignificant for auricular zoster, Ramsay-Hunt syndrome, and melanoma.His family history is notable for a sister with MS. He had been treatedwith AVONEX® since 1998, and experienced three relapses the year beforeenrolling in the placebo-controlled MS study, during which heexperienced no relapses or evidence of progression. He was negative foranti-natalizumab antibodies and his serum concentration of natalizumabwas similar to the mean of the study populations throughout hisparticipation.

In October 2004, his MRI scan showed a small periventricularGd-enhancing lesion on the right and a small right frontal, subcortical,non-enhancing, T2-hyperintense lesion. In November 2004, he exhibitedbehavioral changes followed by hemiparesis and cognitive impairment. Hislast dose of natalizumab was in December 2004. In February 2005, despitetreatment with high dose intravenous methylprednisolone, he continued todeteriorate. A brain MRI scan in February 2005 demonstrated extension ofthe previously identified lesion. He underwent an extensive work-up,including CSF analysis and brain biopsy, which resulted in the diagnosisof PML. Cidofovir treatment was initiated without clinical effect. TheJC viral load decreased in his plasma and CSF over the next few months.This corresponded to further deterioration in his clinical course anddevelopment of Gd-enhancing lesions on MRI, consistent with ImmuneReconstitution Inflammatory Syndrome. He continued to receive treatmentwith cidofovir, and cytarabine was added. Approximately 3 monthsfollowing discontinuation of natalizumab, he began to improve. He isable to converse and can hold high-level conversations about his medicalcourse and treatment, but has significant residual cognitive impairmentwith left hemiparesis and ataxia (Langer-Gould et al., N. Eng. J. Med.353:375-381(2005)).

The final patient was a 60-year-old male with a 28-year history ofCrohn's Disease. Over the course of his illness, he had been treatedwith azathioprine, oral budesonide, corticosteroids, and four doses ofinfliximab. He displayed pre-existing signs of impaired hematopoiesis,predominantly lymphopenia and anemia, since 1996 and receivedazathioprine beginning in 1999. He was enrolled in a Phase 3 study ofnatalizumab in patients with active Crohn's Disease in March 2002 andreceived three doses concomitantly with azathioprine prior to beingrandomized to placebo in a Phase 3 maintenance study. He remained onazathioprine and placebo until November 2002 when azathioprine wasdiscontinued due to refractory pancytopenia. In February 2003, he beganopen-label treatment with natalizumab. He was negative foranti-natalizumab antibodies and his serum concentration of natalizumabwas similar to the mean of the study populations throughout hisparticipation.

In July 2003, one month after his fifth dose of natalizumab, hepresented with a one-week history of cognitive decline. A brain MRI scandemonstrated a large T2-hyperintense lesion in the right frontal lobe,and additional hyperintense lesions in the left frontal and temporallobes that did not enhance with gadolinium. He underwent a partialresection of the lesion, the pathology of which was read at the time asan anaplastic astrocytoma, WHO Grade III. He was treated withcorticosteroids and anticonvulsants, but was too ill for radiationtherapy. Follow-up MRI six weeks after surgery showed tumor extension.He deteriorated clinically and died in December 2003. The case wasreported by the treating physician as a malignant astrocytoma, basedupon the final pathology report. In February, as a result of the oneconfirmed and one suspected case of PML described above, his case wasreassessed and determined to be PML following consultation with twoindependent neuropathologists with expertise in PML (Van Assche et al.,N. Engl. J. Med. 353:362-368 (2005)).

Clinical trial patients exposed to natalizumab were systematicallyassessed for evidence of incipient PML or any other opportunisticinfection. Patients were evaluated if they had any active neurologicaldeterioration for which PML could not be excluded as a diagnosis, showedMRI abnormalities for which PML could not be ruled out, or their CSF haddetectable JCV DNA titers.

Criteria were established prospectively for the neuroradiologic evidenceand laboratory assays for the diagnosis of PML. A diagnosis of“confirmed PML” was defined by presence of progressive clinical disease,MRI signs typical of PML, detection of JCV DNA in CSF, or pathologicconfirmation. Sufficient evidence to exclude PML was defined as lack ofprogressive neurological disease, MRI lesions not typical of PML orstable over time, or no detectable JCV DNA in the CSF if the MRI wassuspicious. A case was deemed “indeterminate” if there was clinical orMRI suspicion of PML and follow-up clinical, MRI, or CSF data could notbe obtained.

A total of 3,826 eligible study participants (2,248 MS patients, and1,578 Crohn's Disease/rheumatoid arthritis patients) were notified toreport to their treating physician/investigators for an assessment.Investigators were requested to perform the assessment procedure,including medical history, neurological examination, brain MRI, and CSFcollection. Blood samples were also collected for PCR analysis of JCVDNA as an exploratory adjunct. MRI scans were assessed by Central ReaderCenters with expertise in neurological disorders, including the twoCentral Reader Centers for the original Phase 3 MS studies. A consensusguideline was developed prospectively to standardize criteria to helpdistinguish MS white matter abnormalities from those of PML.

In all, 3,389 (89%) study patients with MS, Crohn's Disease, orrheumatoid arthritis were assessed by their treating physician, 3,116 ofwhom had received natalizumab. The remaining 273 patients had receivedplacebo as part of a clinical trial and were included as a controlgroup. Of the 437 that were not assessed, 60 (22 MS patients, 38 Crohn'sDisease/rheumatoid arthritis patients) were lost to follow-up. Amongstthe 3,389 patients who participated, 2,046 were MS study patients, over97% of whom were seen within three months of their last natalizumabdose. Six MS patients were referred for further evaluation. Of theseclinical trial patients, five were referred due to neurologicalworsening and one due to possible PML based on MRI findings. MRI scanreview effectively ruled out the diagnosis of PML in the five patientsreferred based on clinical concern. Repeat MRI and CSF analysis excludedPML in the case referred based on MRI findings.

Of the 1,349 Crohn's Disease/rheumatoid arthritis patients whoparticipated in the safety evaluation, 21% were seen within three monthsof their last dose, 91% within six months. Thirty-five patients wereevaluated, including one due to clinical or neurological symptoms, 32based on suspicious changes on MRI, one due to high plasma JCV copynumber, and one due to an inability to perform MRI in a patient with anormal neurological examination. The higher rate of examination ofCrohn's Disease compared to MS was predominantly driven by the lack ofbaseline MRI scans for comparison in the Crohn's Disease population.Most cases were deemed not to be PML based on review of neurologicalexamination, MRI and, if available, CSF testing. For the ten cases inwhich concern still remained, repeat MRI assessments were performed andall were diagnosed as “not PML” based on lack of clinical progression,lack of MRI progression over two months following the initial MRIleading to referral for evaluation, and in some cases, results of CSFtesting.

MRI scans of the brain with and without Gd-enhancement and a FLAIRsequence were sometimes a useful tool for excluding a diagnosis of PMLin the MS cases. The existence of pre-treatment and on-treatment MRIscans increased specificity and assisted in interpretation of thefollow-up MRI scans obtained at varying time points, especially in thesetting when the patient's neurological condition was worsening. Duringthe safety evaluation process, comparison to previous scan was requiredin approximately 35% percent of MS cases because of the presence oflesions for which PML could not be definitely excluded. After comparisonto a prior scan, the neuroradiologist was able to exclude PML in greaterthan 99% of MS cases.

CSF was available for testing in 396 patients who had been treated forMS or Crohn's Disease with natalizumab. JCV was not detected in any ofthese cases, including 19 patients evaluated based on clinical or MRIcriteria. Samples from 411 patients with MS and other neurologicaldisorders served as CSF and plasma controls and were evaluated incollaboration with the Karolinska Institute and the National Institutesof Health (Yousry et al., N. Engl. J. Med. scheduled for publicationMar. 2, 2006). No detectable JCV was found in these CSF samples,confirming the specificity of the CSF assay for only active cases ofPML. Each of the three patients with confirmed PML had detectable JCVDNA. A previous study had indicated that JCV was found in 11% of thebiological specimens of the 121 MS patients tested (Ferrante et al.,Multiple Sclerosis 4:49-54 (1998).

Plasma was tested for the presence of JCV DNA as an exploratory measure.The entire consenting study population (2,370 patients) was evaluatedusing a high-throughput automated system of DNA extraction and PCRanalysis. In addition, a random subset of samples was assessed using amanual low-throughput method. Although the manual method wasdemonstrated to be an order of magnitude more sensitive than thehigh-throughput system, given the techniques involved, testing usingthis method was only possible in approximately 10% of the overallpopulation (209 patients). Of the 2,370 patients from the safetyevaluation who were tested for JC viremia, only five patients (0.2%) haddetectable JCV DNA, three of whom had never received natalizumab. Inaddition, JCV DNA was not detected in any of the 411 samples from MSpatients naïve to treatment and patients with other neurologicaldiseases. These results were confirmed using the manual extractionmethod. In addition, of the random subset of 209 patients tested by themanual method, an additional five (2.4%) samples had detectable JCV DNA.None of the patients with detectable JCV DNA in their plasma by eithermethod had clinical features or MRI findings suggestive of PML.

Serum samples were available from the three patients with confirmed PMLobtained both before and after diagnosis. Only one patient, the patientwith Crohn's Disease, had detectable JCV DNA in the serum prior to theonset of his symptoms. The other two patients had no detectable JCV DNAdespite being clinically symptomatic for the disease and manifestingchanges on a brain MRI scan. The observations in these groups ofpatients are consistent with the data from the literature demonstratingthat the mere presence of JCV DNA in plasma is neither predictive nordiagnostic of PML.

In summary, the comprehensive safety assessment performed following theidentification of PML in natalizumab-treated patients uncovered noadditional confirmed cases of PML in the over 3,000 patients examined.Nearly all patients who had received natalizumab in recent MS, Crohn'sDisease, and rheumatoid arthritis studies were accounted for during theassessments, making it unlikely that any cases of PML were missed. Theoccurrence of PML was limited to two MS cases and one Crohn's Diseasecase, as originally described. The incidence of PML in subjects treatedwith natalizumab in MS and Crohn's Disease clinical trials is thereforeapproximately 1/1,000 with a 95% confidence interval ranging from 0.2 to2.8/1,000. Plasma testing proved to be neither predictive nor diagnosticof PML, consistent with the published literature (Kitamura et al., J.Infect. Dis. 161:1128-1133 (1990); Tornatore et al., Ann. Neurol.31:454-462 (1992); Dorries et al., Virology 198:59-70 (1994); Agostiniet al., J. Clin. Microbiol. 34:159-164 (1996); Dubois et al., AIDS10:353-358 (1996); Knowles et al., J. Med. Virol. 59:474-479 (1999);Dorries et al., J. Neurovirol. 9 (Suppl 1):81-87 (2003)). Clinical andMRI abnormalities were present in two of the three patients with PMLbefore JCV DNA was detected in the plasma. In addition, JCV DNA wasdetected in plasma in several subjects in the study who had no clinicalor radiographic signs of PML, including three who had never receivednatalizumab. These results suggest that establishing one static level ofplasma JCV is not useful in predicting the likelihood of PML inasymptomatic patients. Physicians and patients should remain vigilantfor signs and symptoms of PML and have a low threshold to suspendtreatment and initiate appropriate diagnostic work-up (MRI, CSFanalysis) in natalizumab-treated patients presenting with newneurological decline.

Consequences of Stopping Therapy

The consequences of stopping natalizumab therapy were carefullyevaluated in a Phase 2 study, which involved 213 patients randomized toreceive six monthly infusions of placebo, 3 mg/kg natalizumab, or 6mg/kg natalizumab. Patients were followed for seven months after thelast infusion. During that time, relapses and other adverse events wererecorded, and MRI scans were performed four months and seven monthsafter the last dose of natalizumab. Comparisons were made between theplacebo group and the two natalizumab dosing groups. As expected, theproportion of patients experiencing relapse, as well as the frequency ofrelapses, rose in the natalizumab group to levels comparable to those inthe placebo group after the cessation of study drug. Moreover, there wasa gradual rise in the proportion of active MRI scans in the natalizumabgroup to levels comparable to that of the placebo group after thecessation of therapy. Thus, the cessation of natalizumab treatmentresulted in loss of efficacy, but there was no evidence of an increasein disease activity beyond that which would have been expected had therebeen no treatment with natalizumab, i.e., no rebound effect wasobserved. Therefore, MS patients who discontinue natalizumab therapy donot have an increased risk for marked increase in disease activity.

Drug Interactions

In a placebo-controlled MS study, the administration of AVONEX® appearedto be associated with an increase in the serum concentrations ofnatalizumab in a small cohort on whom intensive pharmacokinetic samplingwas performed. However, based upon a comparison of the mean post-hocparameter estimates from the population pharmacokinetic analysis,steady-state clearance and half-life values differed between patientsconcurrently taking AVONEX® and natalizumab monotherapy, but only byapproximately 5%, and were not considered clinically significant. Inaddition, natalizumab was well tolerated when administered to 589patients in combination with AVONEX® for up to 120 weeks. It is notablethat the two reports of PML in the MS database occurred in patientsreceiving concomitant AVONEX®. Thus, the risk of PML with natalizumabtreatment may be increased by concomitant treatment with interferon β,though this could have occurred in two patients on combination therapydue to chance alone (p=0.23).

The safety of natalizumab in combination with glatiramer acetate wasevaluated by administering natalizumab over six months to patients whocontinued to receive 20 mg of daily glatiramer acetate. There were nointeractions between glatiramer acetate and natalizumab pharmacokineticsor its α4-integrin receptor saturation. However, this study was ofinsufficient size or duration to establish the long-term safety orefficacy in this population.

Efficacy of Natalizumab

Multiple Sclerosis

MS is a chronic disease of the brain and spinal cord. In temperate zonessuch as the United States, the incidence of MS is approximately 1 to5/100,000 per year (US National MS Society; NMSS), with a US prevalenceestimated at 350,000 to 400,000. It is a disease of young adults,primarily women, with disease onset typically occurring between the agesof 20 and 40. The first clinical manifestations of MS usually take theform of a clinically isolated syndrome affecting the optic nerve (opticneuritis), spinal cord (transverse myelitis), or brainstem/cerebellum(Runmarker and Anderson, Brain 116:117-134 (1993)). Estimates of thenumber of patients who eventually go on to develop MS vary widely, but,in the case of optic neuritis, the presence of MS-like lesions on MRI atthe time of the attack indicates a greater than 80% chance of developingclinically definite MS within 10 years (O'Riordan et al., Brain121:495-503 (1998); Sailer et al., Neurology 52:599-606 (1999)).

Demyelination and nerve fiber transection is thought to occur whenactivated T lymphocytes cross the blood-brain barrier and initiate aseries of events leading to activation of endothelial cells, recruitmentof additional lymphocytes and monocytes, and release of pro-inflammatorycytokines. MS lesions typically consist of immune cells, demyelinatedaxons, oligodendrocytes attempting remyelination, proliferatingastrocytes, and varying degrees of axonal transection. Cytokines such astumor necrosis factor-alpha (TNF-α) and interferon gamma (IFN-γ)interact with immune cells, amplifying this process. The initiatingevent of the inflammatory cascade is unknown; however, adhesion andtrans-endothelial migration of inflammatory cells from the bloodstreamacross the blood-brain barrier and into the central nervous system (CNS)is thought to be an early and critical step in this process.

Emerging data demonstrate that irreversible axonal loss occurs early inthe course of MS. Because transected axons fail to regenerate in theCNS, early effective treatment aimed at suppressing MS lesion formationis of paramount importance. As early as disease onset, axons aretransected in lesions with active inflammation (Trapp et al., N. Engl.J. Med. 338:278-285 (1998); Bjartmar and Trapp, Curr. Opin. Neurol.14:271-278 (2001); Ferguson et al., Brain 120 (Pt 3):393-399 (1997)).The degree of demyelination is related to the degree of inflammation andthe exposure of demyelinated axons to the inflammatory environment, aswell as non-inflammatory mediators (Trapp et al., N. Engl. J. Med.;338:278-285 (1998); Komek et al., Am. J. Pathol. 157:267-276 (2000);Bitsch et al., Brain 123:1174-1183 (2000)). There is also destruction ofoligodendrocytes with impaired remyelination in demyelinating lesions(Peterson et al., J. Neuropathy Exp. Neurol. 61:539-546 (2002); Chang etal., J. Neurovirol. 8:447-451 (2002)). The loss of oligodendrocytesleads to a reduction in the capacity to remyelinate and may result inthe loss of trophic factors that support neurons and axons (Bjartmar etal., J. Neurocytol. 28:383-395 (1999)).

The typical inflammatory lesions of MS can occur throughout the CNS, butcertain sites seem particularly vulnerable, such as the optic nerve,brainstem, spinal cord, and periventricular regions of the cerebrum. Itis the resulting loss of myelin and nerve fibers in these areas thatleads to impaired neuronal conduction and symptoms such as weakness,sensory loss, visual loss, double vision, and imbalance. In relapsingremitting MS, these episodes of demyelination typically result inseveral weeks of neurological dysfunction followed by partial or fullrecovery. However, more severe attacks may result in permanent deficits.The recurrent attacks over time lead to accumulating physical disabilityand cognitive decline.

A number of measures, including clinical measures, those based on MRIscans, and those based on quality of life, can be used to assess aproduct's efficacy in treating MS. The Expanded Disability Status Scale(EDSS) is an extensively used tool for tracking the course of disabilityin MS. It classifies the most common MS-associated neurologicalimpairments into disability levels ranging from 0 to 10, with eachsuccessive step describing a worsening of disease. In the lower range ofthe EDSS scale, disease progression is primarily defined by increasinglevels of disability in specific functional systems measured duringneurological examination. Scores of 1.0 through 3.5 describe mild tomoderate disability in the functional systems. Higher scores, in therange of 4.0 and above indicate increasingly severe disability thataffects ambulation, including the need for assistive devices such as acane (an EDSS of 6.0), a walker (an EDSS of 6.5), or a wheelchair (anEDSS of 7.0). Scores higher than 7.0 classify patients confined to bed.

The MS Functional Composite (MSFC) (Whitaker et al., Multiple Sclerosis1:37-47 (1995)) is also used to assess efficacy. Unlike traditional MSclinical outcome measures that are derived from the standardneurological examination, the MSFC is based on quantitative tests of legfunction/ambulation (the Timed 25-Foot Walk), arm function (theNine-Hole Peg Test), and cognitive function (the Paced Auditory SerialAddition Test (PASAT 3)) which expand upon the measurements of the EDSSand assess effects in clinical dimensions not well captured by thisscale.

MRI is another tool for assessing efficacy in treating MS and can beused alone or to support clinical data to assess therapeutic effects onrelapse and disability endpoints. MRI is a sensitive tool for monitoringdisease activity, detecting approximately five to ten times more diseaseactivity in both relapsing remitting MS and secondary progressive MSpatients than is clinically apparent (Isaac et al., Neurology38:1511-1515 (1988); Willoughby et al., Ann. Neurol. 25:43-44 (1989);Khoury et al., Neurology 44:2120-2124 (1994); Thompson et al., Ann.Neurol. 9:53-62 (1991); Thompson et al., Neurology 42:60-63 (1992)).T2-weighted sequences in MS patients detect new areas of acutedemyelination, as well as more chronic areas of demyelination andgliosis. For this reason, T2-weighted MRI is a good technique formonitoring the accumulation of lesions over time, either as a count ofactive lesions or a change in the total volume of such lesions.

Infusion of gadolinium-diethylenetriamine pentaacetic acid (Gd-DPTA)during acquisition of T1-weighted sequences allows for visualization ofblood-brain barrier breakdown secondary to the inflammationcharacteristic of acute MS lesions. The evidence to date suggests thatgadolinium (Gd)-enhancement is a useful marker of disease activity thatcorrelates with clinical relapse (Molyneux et al., Ann. Neurol.43:332-339 (1998); Kappos et al., Lancet 353:964-969 (1999); McFarlandet al., Multiple Sclerosis 8:40-51 (2002)).

New hypointense lesions on T1-weighted sequences in MS patientscorrespond either with inflammatory Gd-enhancing lesions (comprisingedema, demyelination, axonal loss, or combinations of these pathologies)(Bruck et al., Ann. Neurol. 42:783-793 (1997)) or as chronic lesionswith considerable axonal loss. Approximately half of the acute T1hypointensities on MRI will evolve into chronic “T1 black holes,” whichcorrelate with disability progression (Simon et al., Neurology55:185-192 (2000)).

As described in more detail in Example 1, two Phase 3 studies wereconducted to study the effect of two years of treatment withnatalizumab. One of the studies used natalizumab alone (the monotherapystudy) and the other used natalizumab in combination with AVONEX® (theadd-on therapy study). Both these Phase 3 studies were designed with twosets of primary and secondary endpoints. The primary and secondaryendpoints were selected to measure the effects of natalizumab on theinflammatory aspects of the disease after a mean of one year offollow-up in each study (900 patient-years of observation in themonotherapy study; 1,200 patient-years in the add-on therapy study).

The primary endpoint of these studies was the annualized rate ofclinical relapses. Two of the secondary endpoints were two supportingMRI measures of inflammatory disease activity, namely, the mean numberof new or newly enlarging T2-hyperintense lesions (measuring lesionaccumulation over time) and the mean number of Gd-enhancing lesions(measuring acute disease activity), as ranked in order of importance.The proportion of patients remaining relapse-free provided a thirdsecondary endpoint.

Another series of endpoints was assessed at the conclusion of each studyfollowing two years of natalizumab treatment. The endpoints for thisfinal analysis were selected to determine natalizumab's effects onmeasures associated with MS disease progression. The primary endpoint attwo years was the time until onset of sustained progression ofdisability, as measured by changes in EDSS scores. Similar to theone-year analysis, the secondary endpoints were additional MRI andclinical measures that would support the primary analysis. The secondaryendpoints at two years, ranked in order of importance, were the rate ofMS relapses (to confirm one-year relapse observations), the mean volumeof T2-hyperintense lesions (a measure of overall MS disease burden), themean number of T1-hypointense lesions (a measure of axonal loss), andprogression of disability as determined by changes in the MSFC (toconfirm and expand upon disability effects as measured by the EDSS).

Given two primary endpoints at two different time points (annualizedrelapse rate at one year, time to disability progression at two years),the Hochberg procedure for multiple comparisons (Hochberg, Biometrika75:800-802 (1988)) was used to evaluate the primary endpoint. Each setof secondary endpoints was prioritized in order of importance as listedabove. A closed testing procedure was used for each set, such that ifstatistical significance was not achieved for an endpoint within a set,all endpoints(s) of a lower rank in that set were not consideredstatistically significant. Analyses of tertiary endpoints did notinclude adjustments for multiple comparisons.

Monotherapy with Natalizumab

These results of the monotherapy study indicated that natalizumab is aneffective treatment as monotherapy for relapsing remitting MS.Natalizumab treatment resulted in significant effects on relapse rates,disability progression, and all MRI measures, the primary and secondaryendpoints of the study. Analysis of Kaplan-Meier curves indicate thatthe impact on relapse rates and disability progression was apparentearly after treatment initiation, and was sustained throughout thetreatment period with patient groups continuing to diverge at the finaltimepoint. Further, these findings were consistent across subgroups.Additional positive effects were seen on measures of relapse severityand quality of life.

MS patients treated with natalizumab alone had a 42% lower risk of theirdisability progressing compared to placebo, as measured by changes onthe EDSS, the primary endpoint of the study at two years (p<0.001). Thepercentage of patients estimated to progress was 17% and 29% withnatalizumab and placebo, respectively. In addition to the EDSS,natalizumab had significant effects on all relapse endpoints studiedover two years, including a 68% reduction in the annualized relapse ratecompared to placebo, with 67% of natalizumab-treated patients remainingrelapse-free, compared to 41% of patients on placebo. The MRI scanssupported these clinically-observed effects. Also, natalizumab treatmentimproved the patients' quality of life, as measured by the physical andmental components of the SF-36. All these effects were consistent andsignificant across subgroups defined by baseline demographics anddisease activity.

Combination Therapy of Natalizumab and AVONEX®

A significant number of patients who receive the currently approvedtherapies continue to experience disease activity, as measured bothclinically and by MRI. This is an expected outcome of these partiallyeffective approved medications, each of which leads to an approximately30% reduction in relapse rate (IFNB MS Study Group, Neurology 43:655-661(1993); Jacobs et al., Ann. Neurol. 39:285-289 (1996); PRISMS StudyGroup, Lancet 352:1498-1504 (1998); Johnson et al., Neurology45:1268-1276 (1995)). Data from the Phase 3 trials of β-interferon forthe treatment of MS show that 62% to 75% of patients experienced atleast one relapse during these two-year trials despite interferontreatment IFNB MS Study Group, Neurology 43:655-661 (1993); Jacobs etal., Ann. Neurol. 39:285-289 (1996); PRISMS Study Group, Lancet352:1498-1504 (1998)). Similarly, 66% of subjects in the Phase 3 MStrial of glatiramer acetate experienced at least one relapse during the2-year period, a number that was not significantly different fromplacebo (Johnson et al., Neurology 45:1268-1276 (1995)). Although avariety of therapeutic strategies are currently in use in clinicalpractice to manage breakthrough disease while on treatment (e.g.,switching therapy, changing dose and frequency of interferon,combination therapy), these practices are largely empirical as there areno randomized, controlled trials to assess the efficacy of theseapproaches.

The add-on therapy study was designed to evaluate the efficacy ofnatalizumab against active control for patients breaking through AVONEX®monotherapy. The choice of β-interferon was supported by available dataon the proposed mechanisms of action of the available drugs. Asdiscussed above, natalizumab has a well-defined mechanism of action,specifically targeting cellular adhesion and trans-endothelial migrationvia α4-integrins. Although the exact mechanism by which interferon-βexerts efficacy in MS is not known, interferon-β induces a large numberof cellular processes involved in cytokine secretion and cellularphenotype changes. Interferon-β down regulates interferon-γ induced MHCclass II molecule production, decreases secretion of TH1pro-inflammatory cytokines (TNF-α, IL-2 and interferon-γ) and increasessecretion of TH2 anti-inflammatory cytokines (IL-4 and IL-10) (Rep etal., J. Neuroimmunol. 67:111-118 (1996); Kozovska et al., Neurology53:1692-1697 (1999); Rudick et al., Neurology 50:1266-1272 (1998)). Inaddition, interferon-β may affect leukocyte trafficking throughsuppression of the chemokines RANTES and MIP-1α, as well as theirreceptor CCR5 (Zang et al., J. Neuroimmunol. 112:174-180 (2001)). Thereis, therefore, scientific rationale to expect that the blockade ofα4-integrins by natalizumab, when added to interferon-β, may have anadditive or synergistic effect when added to interferon-β alone.

Natalizumab was also proven efficacious when used to treat patientsconcurrently receiving treatment with AVONEX®. Prior to receivingnatalizumab, these patients were experiencing disease activity despiteactive treatment. Thus, AVONEX® served as an active control. The studydemonstrated that natalizumab, when added to AVONEX®, resulted in a 24%reduction in the risk of disability progression, as measured by changeson the EDSS (p=0.024). The percentage of patients estimated to progresswas 23% with natalizumab plus AVONEX® as compared with 29% on AVONEX®alone.

Natalizumab had significant effects on all relapse endpoints examined,when compared to AVONEX® over two years, including a 55% reduction inthe annualized relapse rate, with 54% of natalizumab-treated patientsrelapse-free compared to 32% of patients on AVONEX®. The MRI scanssupported these clinically-observed effects. Also, natalizumab, whencompared to AVONEX® therapy alone, improved the patients' quality oflife, as measured by the physical components of the SF-36, with a trendon the mental component. All these effects were consistent andsignificant across subgroups defined by baseline demographics anddisease activity.

Progressive Multifocal Leukoencephalopathy

PML is an infectious disease of the central nervous system caused by JCVinfection of oligodendrocytes. JCV is a human polyoma virus that isbelieved to infect the majority of healthy individuals at an early age.The seroprevalence of anti-JCV antibodies in healthy individuals hasbeen estimated to range from 20% to 80% depending upon the testingmethodology (Knowles et al., J. Med. Virol. 71:115-123 (2003)); Knowlesand Sasnauskas, J. Virol. Methods. 109:47-54 (2003)).

PML occurs predominantly in immunocompromised individuals with anage-adjusted death rate due to PML of 3.3 per million persons (in 1994),89% of whom were AIDS patients (Holman et al., Neuroepidemiol.17:303-309 (1998)). However, rare PML cases have also been reported inpatients with autoimmune disorders who received immunosuppressivetherapy; among these, three patients with rheumatoid arthritis(Sponzilli et al., Neurology 25:664-668 (1975); Rankin et al., J.Rheumatol 22:777-79 (1995); Durez et al., Arthritis Rheum. 46 (9S):536(2002)), one of whom was treated with tumor necrosis factor (TNF)antagonist (Durez et al., Arthritis Rheum. 46 (9S):536 (2002)). Therewas also a report of PML in a Crohn's Disease patient, but theconcomitant treatments were not specified (Garrels et al., Am. J.Neuroradiol. 17:597-600 (1996)).

The pathology of PML is distinctive and comprises multiple foci ofdemyelination of varying size from pinpoint lesions to areas of severalcentimeters. The lesions may occur anywhere but are usually in thecerebral hemispheres, less often in the cerebellum and brain stem andrarely in the spinal cord. The oligodendrocytes in the peripheral zonesurrounding an area of demyelination are grossly abnormal. The nuclei ofabnormal oligodendrocytes are packed with JC virions. Typically, PMLevolves gradually, with impairment of mental function and disturbance ofspeech and vision. Movement may also be affected. The disease thenprogresses rapidly and the patient is severely disabled, eventuallybecoming demented, blind, and paralyzed; coma and death follow.

The presence of JCV in the blood and urine of PML patients and healthy,immunocompetent individuals has been described (Kitamura et al., J.Infect. Dis. 161:1128-1133 (1990); Tornatore et al., Ann. Neurol.31:454-462 (1992); Dorries et al., Virology 198:59-70 (1994); Sundsfjordet al., J. Infect. Dis. 169:485-490 (1994); Agostini et al., J. Clin.Microbiol. 34:159-164 (1996); Dubois et al., AIDS 10:353-358 (1996);Knowles et al., J. Med. Virol. 59:474-479 (1999); Dorries et al., J.Neurovirol. 9(Suppl 1):81-87 (2003)). These findings are neitherpredictive nor diagnostic of PML in these patients; thus therelationship of blood or urine viral load to PML is unclear.

The clinical presentation of PML is largely dependent upon the size anddistribution of the white matter lesions that develop as a result ofviral infection, demyelination, and glial cell lysis. However, clinicalfeatures of the presentation help differentiate it from thedemyelination associated with MS. In contrast to MS, PML involvement ofthe spinal cord or optic nerves is rare. Instead, about one-third ofpatients will present with visual field loss or cortical blindness withanother third presenting with altered mentation or behavior changes(Dworkin et al., Curr. Clin. Top. Infect. Dis. 22:181-195 (2002)). Alsounlike MS, hemiparesis is a common presenting symptom. These symptomsare typically subacute in onset and follow a slowly progressive course.Often, patients and their families are the first to notice the onset ofPML through changes in the ability to perform routine activities ofdaily living, even prior to presentation with changes on neurologicalexamination.

MRI is a sensitive tool for the detection of PML lesions in the settingof clinical signs or symptoms, although it may lack specificity. TypicalMS lesions, demyelination from other causes (e.g., encephalomyelitis,HIV encephalopathy), gliosis, and edema can often have an appearancesimilar to early PML lesions. However, as shown in Table 1, there arefeatures of PML lesions that help differentiate them from otheretiologies (Post et al., Am. J. Neuroradiol. 20:1896-1906 (1999); Yousryet al. N. Engl. J. Med. in press (2006); (Berger et al., Ann. Neurol.44:341-349 (1998); Hoffmann et al., J. Neurol. Neurosurg. Psychiatry74:1142-1144 (2003); Langer-Gould et al., N. Engl. J. Med. 353:375-381(2005)).

TABLE 2 Differential Diagnosis of MS and PML MS PML Location of newlesions Mostly focal, may affect entire brain and Diffuse, mainlysub-cortical, rarely spinal cord, in white and possibly grayperiventricular, almost exclusively in matter; white matter, althoughoccasional Posterior fossa lesions rarely seen extension to gray matterseen; Posterior fossa frequently involved (cerebellum) Borders Sharpedges, shapes mostly round or Ill-defined edges, infiltrating, irregularin finger-like (especially periventricular), shape, confined to whitematter, sparing confluent with other single lesions, U- gray matter,pushing against cortex, fibers may be involved U-fibers destroyed Modeof extension Focal, enlarging of lesions within Diffuse, asymmetrical,extending days/weeks, later decreasing in size within homogeneously, noconfluence with other months lesions, defined to white matter tracks,sparing cortex, continuous progression Mass effect Acute lesions mayshow some mass effect No mass effect even in large lesions (but processis slightly pushing against cortex) T2-weighted sequence Acute lesions:hyperintense center, Diffuse hyperintense, slightly increased isointensering, discrete hyperintensity intensity of newly involved areas outsidering structure; compared to old areas, little irregularSub-acute/chronic lesions: hyperintense, no signal intensity of lesionsring structure T1-weighted sequence Acute lesions: densely hypointense(large Slightly hypointense from the onset, lesion) or isointense (smalllesion), signal intensity decreasing over time and increasing signalintensity over time in along the affected area, no reversion of 80%,decreasing signal intensity (axonal signal intensity loss) in about 20%Flair sequence Hyperintense, sharply delineated Hyperintensity moreobvious, true extension of abnormality more clearly visible than inT2-weighted images Enhancement Acute lesions: dense homogeneous Usuallyno enhancement even in large enhancement, sharp edges lesions, in HIV+patients some peripheral Sub-acute lesions: ring-enhancement enhancementpossible, especially under Chronic lesions: no enhancement therapyAtrophy Focal atrophy possible due to focal white No focal atrophy sinceextending matter degeneration, no progression pathological process isslightly pushing against cortex (extension of tissue)

MRI analysis can provide a differential diagnosis of MS and PML inpatients receiving natalizumab. Patients suspected of PML demonstratethe presence of multifocal, asymmetric, white-matter lesions reflectiveof demyelination by MRI. T₂-weighted and fluid-attenuated inversionrecovery (FLAIR) MRI reveals hyperintense lesions throughout thesupratentorial subcortical white matter (Post et al., Am. J.Neuroradiol. 20:1896-1906 (1999)). White matter lesions of PML aretypically not surrounded by edema, do not produce a mass effect, and donot enhance in the presence of gadolinium contrast material (Post etal., Am. J. Neuroradiol. 20:1896-1906 (1999)). However, hyperintenseT₂-weighted and FLAIR images are not specific for demyelination and mayrepresent gliosis or edema. Other demyelinating, encephalopathic orischemic processes such as MS, postviral encephalitis, HIVencephalopathy and infarction, may demonstrate similar non-specificimaging features (Olsen et al., Radiology 169:445-448 (1988), Hurley etal., J. Neuropsychiatry Clin. Neurosci. 15:1-6 (2003)). The location oflesions and their morphological characteristics, the absence or anatypical presence of gadolinium enhancement on T₁-weighted images, andthe implementation of magnetization transfer MRI may also helpdifferentiate the demyelination of PML from other demyelinatingprocesses, edema or gliosis (Ernst et al., Radiology 210:439-543 (1999);Hurley et al., J. Neuropsychiatry Clin. Neurosci. 15:1-6 (2003)).

The clinical diagnosis of PML is confirmed by histological andvirological examination of brain material obtained by brain biopsy or atpostmortem. Before a biopsy is done, both serum and CSF should beexamined for antibodies against JCV. A positive result will not confirmPML, but a negative result makes the diagnosis of PML very unlikely. Itis rare to detect antibodies against JC in the CSF, and when they aredetected, it is suggestive of active multiplication of JCV within theCNS. The brain biopsy or autopsy material can be examined by electronmicroscopy or immunohistologic electron microscopy. The specimen canalso be examined directly for JCV antigen by immunofluorescence orimmunoperoxidase staining. Viral isolation of JCV has been reported tobe difficult, but may be attempted from primary human fetal glial cells.The presence of the virus in culture is confirmed by electronmicroscopy, immunofluorescence, or haemagglutination.

PCR analysis of the CSF for JC viral DNA is a highly sensitive andspecific test for the diagnosis of PML. The specificity of this testapproaches 100%, with a sensitivity ranging from 60% to 90% (Henson etal., Neurology 41:1967-1971 (1991); Gibson et al., J. Med. Virol.39:278-281 (1993); Weber et al., AIDS 8:49-57 (1994a); Weber et al. J.Infect. Dis. 169:1138-1141 (1994b); Vago et al., J. Acquir. Imm. Defic.Syndr. Hum. Retrovirol. 12:139-146 (1996)). In cases with a highclinical suspicion of PML and negative CSF results, repeat testing oftenleads to detection of JC viral DNA. As such, PCR analysis of the CSF forJC viral DNA has grown to be the preferred method to confirm thediagnosis of PML.

Untreated, PML patients have a mortality rate of 30% to 50% during thefirst three months (Koralnik, Curr. Opt. Neurol. 17:365-370 (2004)).Prior to the introduction of highly active antiretroviral treatment(HAART) for HIV, about 10% of patients with PML survived for longer thanone year. However, since the advent of HAART, about 50% of patients withPML survive for longer than one year due to restoration of immunefunction as CD4 counts increased as a result of immune reconstitutioninflammatory syndrome (Geschwind et al., J. Neurovirol. 7:353-357(2001); Berger et al., Ann. Neurol. 44:341-349 (1998); Clifford et al.,Neurology 52:623-625 (1999); Tantisiriwat et al., Clin. Infect. Dis.28:1152-1154 (1999)).

Currently, there is no established drug treatment for PML. Variousmedications have been tested, including acyclovir, idoxuridine,vidarabine, amantadine, adenine arabinoside, cytosine arabinoside(cytarabine), cidofovir, interferon α, interleukin-2 (IL-2), zidovudine,camptothecin, and topotecan (Koralnik, Curr. Opt. Neurol. 17:365-370(2004); Dworkin et al., Curr. Clin. Top. Infect. Dis. 22:181-195 (2002);Seth et al., J. Neurovirol. 9:236-246 (2003); Collazos, CNS Drugs17:869-887 (2003); Mamidi et al., J. Neurovirol. 8:158-167 (2002);Przepiorka et al., Bone Marrow Transplant; 20:983-987 (1997); Redingtonet al., Arch. Neurol. 59:712-718 (2002); Padgett et al., Prog. Clin.Biol. Res. 105:107-117 (1983)). However, the survival of patients withPML appears to be best correlated with immune reconstitution. Intransplant patients with PML, early dosage reduction or/anddiscontinuation of immunosuppressive therapy was associated withfavorable clinical outcome after PML diagnosis (Crowder et al., Am. J.Transplant 5:1151-1158 (2005); shitrit et al., Transpl. Int. 17:658-665(2005)).

JC Virus (JCV)

JCV is a member of the class of human polyomavirus, which belong to thePapovaviridae family of small, nonenveloped viruses with a closed,circular double DNA-stranded genome. Polyomaviruses can be distinguishedfrom papillomaviruses by virtue of their smaller virion size anddifferent genomic size and organization. Polyomaviruses are ubiquitousin nature and can be isolated from a number of species. JCV was firstisolated from the brain tissue of a patient with progressive multifocalleukoencephalopathy (PML). JCV shares 75% nucleotide sequence homologywith the BK human polyomavirus (BKV), which was isolated from the urineof a renal transplant patient with postoperative ureteral stenosis. BKVand JCV each share 70% homology with SV40. The two are not serologicallycross-reactive and serologic tests for antibodies are able todistinguish between BKV and JCV (Demeter, in Mandell et al., eds.,Mandell, Douglas and Bennett's Principles and Practice of InfectiousDiseases, 4th edition, Vol. 2. New York, N.Y.: Churchill Livingstone;1995:1400-1406).

JCV infection is usually sub-clinical, is almost universal, occurs inchildhood, and persists for life. It is estimated that 60-80% of adultsin Europe and the United States have antibodies to JCV and that 50% ofyoung adults in the age range of 30-39 years have been infected withJCV. JCV and BKV are believed to circulate independently. It has beenproposed that JCV establishes latent infections in the kidney and/or theCNS after a primary infection (Demeter, in Mandell et al., eds.,Mandell, Douglas and Bennett's Principles and Practice of InfectiousDiseases, 4th edition, Vol. 2. New York, N.Y.: Churchill Livingstone;1995:1400-1406). During immunosuppression, it has been postulated thatlatent JCV is reactivated in the kidney, which may lead to viruria.While viruria may have some predictive value for PML, since it does notoccur in the majority of PML cases, measuring JCV in the urine alone isnot sufficient to diagnose JCV.

When JCV travels through the bloodstream to the brain, it may attackmyelin-producing cells. The resulting brain infection producesneurological symptoms which may include ataxia, loss of cognitivefunction, visual loss, changes in balance and coordination, and loss ofsensation. Death commonly occurs within two years following diagnosis.

No specific antiviral therapy that has been proven effective for JCV,and current treatment of immunocompromised patients is primarilysupportive and intended to reduce immunosuppression. Cidofovir iscurrently being studied as a treatment option for transplant patients,and cytarabine can be used in the treatment of PML, although there iscurrently conflicting data regarding the efficacy of the latter(Demeter, in Mandell et al., eds., Mandell, Douglas and Bennett'sPrinciples and Practice of Infectious Diseases, 4th edition, Vol. 2. NewYork, N.Y.: Churchill Livingstone; 1995:1400-1406; Salmaggi, Neurol.Sci. 22:17-20 (2001)).

The cellular receptor for JCV has been reported to be the serotonin5HT2(A) receptor (Elphick et al., Science 306:1380-1383 (2004)). Invitro, the antipsychotic medications chlorpromazine and clozapine wereshown to block the serotonin 5HT2(A) receptor and to block JCV cellentry. Unfortunately, however, chlorpromazine and clozapine have suchsignificant side effects and toxicities, e.g., extrapyramidal symptomsand the possibility of bone marrow dyscrasias that they may beproblematic to use clinically. The invention provides that neweratypical antipsychotics, such as zisprasidone, risperidone, andolanzapine—medicines with much better side effect and toxicity profilesthan the older antipsychotics—are significantly more potent 5HT2(A)receptor antagonists in vitro than chlorpromazine and clozapine.

A wide variety of serological tests are available to detect JCV, e.g.,complement-fixation (CFT), haemagglutination-inhibition (HAI),enzyme-linked immunoassay (EIA), radioimmunoassay (RIA), particleagglutination, immunofluorescence (IF), single radial hemolysis, andWestern blot. The sensitivity and specificity varies greatly betweendifferent techniques. Most techniques will detect all classes ofantibody, whereas some assays e.g., RIA, EIA, and IF can be designed todetect one specific class, for example, IgM, IgG, or IgA.

Patient Selection Based on Safety and Efficacy

Appropriate patient selection helps maximize the benefit-risk profile ofnatalizumab. Natalizumab has demonstrated efficacy in treatment-naïvepatients with mild to moderate disability (EDSS 0 to 5.0) with recentclinical disease activity (for example, one relapse in the year prior tostudy entry). It has also demonstrated efficacy in patients with mild tomoderate disability with continuing disease activity despite treatmentwith β-interferon (for example, one relapse in the year prior to studyentry, while receiving AVONEX®).

The benefit/risk ratio is altered in certain other patient populations.Patients without evidence of relapsing disease, that is, withoutevidence of inflammatory activity clinically or by MRI, such as thosewith relatively “benign” inactive disease, or chronic-progressive formsof MS, were excluded from the Phase 3 trials, thus, natalizumab has notbeen completely evaluated in these cohorts. The benefit-risk is alsoaltered in patients with a single clinical event without featuressuggestive of MS.

Patients who are clinically stable on current therapy also have analtered benefit/risk ratio. If safety or tolerability concerns exist onthe current treatment, or imaging studies indicate active inflammatorysub-clinical disease, natalizumab treatment would be appropriate. Inconsidering the benefit-risk ratio, it should be considered whether thepatient has previously suffered a hypersensitivity reaction or developedpersistent antibodies to natalizumab. Re-dosing of natalizumab followinga hypersensitivity reaction was not assessed in Phase 3 trials.Persistent antibodies against natalizumab lead to a loss of efficacy andan increase in infusion-related side effects. Patients who areimmunocompromised from any cause, including use of immunosuppressantmedications have an independent risk factor for PML and otheropportunistic infections and should not receive natalizumab.

Another criteria for patient selection is a pre-infusion checklist usedby the infusion nurse to facilitate early detection of PML and minimizeinappropriate use of natalizumab. The checklist prompts the nurse to askthe patient about continuously worsening neurological symptoms that havepersisted over several days, e.g., new or sudden decline in thinking,eyesight, balance, or strength. If a patient reports having any symptomsdescribed by the checklist, the nurse is instructed not to administernatalizumab and to refer the patient to his or her physician.

This checklist also ascertains that the patient will be receivingnatalizumab for the treatment of relapsing MS, has never been diagnosedwith PML, and is not currently experiencing any continuously worseningsymptoms that have persisted over several days. It further ascertainsthat the patient is not known to be suffering from HIV or a hematologicmalignancy, nor has had an organ transplant. It confirms that thepatient is not currently receiving treatment with an anti-neoplastic,immunomodulatory, or immunosuppressive agent and that the patient hasread the natalizumab patient information leaflet.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. Moreover, advantages described in the body of thespecification, if not included in the claims, are not per se limitationsto the claimed invention.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Moreover, it mustbe understood that the invention is not limited to the particularembodiments described, as such may, of course, vary. Further, theterminology used to describe particular embodiments is not intended tobe limiting, since the scope of the present invention will be limitedonly by its claims. The claims do not encompass embodiments in thepublic domain.

With respect to ranges of values, the invention encompasses eachintervening value between the upper and lower limits of the range to atleast a tenth of the lower limit's unit, unless the context clearlyindicates otherwise. Further, the invention encompasses any other statedintervening values. Moreover, the invention also encompasses rangesexcluding either or both of the upper and lower limits of the range,unless specifically excluded from the stated range.

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of ordinary skillin the art to which this invention belongs. One of ordinary skill in theart will also appreciate that any methods and materials similar orequivalent to those described herein can also be used to practice ortest the invention. The specification is most thoroughly understood inlight of the references cited herein.

It must be noted that, as used herein and in the appended claims, thesingular forms “a,” “or,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “asubject polypeptide” includes a plurality of such polypeptides andreference to “the agent” includes reference to one or more agents andequivalents thereof known to those skilled in the art, and so forth.

Further, all numbers expressing quantities of ingredients, reactionconditions, % purity, polypeptide and polynucleotide lengths, and soforth, used in the specification and claims, are modified by the term“about,” unless otherwise indicated. Accordingly, the numericalparameters set forth in the specification and claims are approximationsthat may vary depending upon the desired properties of the presentinvention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits, applying ordinary roundingtechniques. Nonetheless, the numerical values set forth in the specificexamples are reported as precisely as possible. Any numerical value,however, inherently contains certain errors from the standard deviationof its experimental measurement.

EXAMPLES

The examples, which are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway, also describe and detail aspects and embodiments of the inventiondiscussed above. The examples are not intended to represent that theexperiments below are all or the only experiments performed. Effortshave been made to ensure accuracy with respect to numbers used (forexample, amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in degrees Centigrade, and pressure is at or nearatmospheric.

Example 1: Efficacy of Natalizumab

The efficacy of natalizumab over a two year period has been demonstratedin two Phase 3 trials (Polman et al., N. Engl. J. Med. in press (2006);Rudick et al. N. Engl. J. Med. in press (2006)). In one study,natalizumab was given as monotherapy to treatment-naïve MS patients andits efficacy was compared to placebo. In the other study, natalizumabwas given to patients who were experiencing relapses despite concurrentAVONEX® therapy and its efficacy was compared to that of AVONEX(interferon β-1a) plus placebo. Data through two years have confirmedthe benefit that led to accelerated approval at one year. These datashow that natalizumab is highly efficacious in delaying the time toonset of sustained progression of disability, in reducing annualizedrelapse rate, in attenuating MRI lesions, and in improving the qualityof life of patients compared both to placebo and the active AVONEX®control group.

Both Phase 3 studies had similar designs. In the monotherapy study, 942untreated relapsing remitting MS patients were randomized to receivenatalizumab or placebo for 120 weeks (30 infusions) using a 2:1allocation. In the add-on study, 1,171 patients who had been receivingweekly intramuscular injections of 30 μg AVONEX®, but who had relapseddespite this treatment, were randomized using a 1:1 allocation to addnatalizumab or placebo to their regimen, also for 120 weeks.

Efficacy parameters included EDSS scores, MS relapses, brain MRI scans,MSFC scores, visual function tests, and quality of life. EDSS and MSFCwere measured every 12 weeks, brain MRI scans and quality of lifequestionnaires at baseline and every year, and MS relapses on an ongoingbasis.

Treatment with natalizumab as monotherapy in treatment-naïve patientshad profound effects on the time to onset of sustained progression indisability and on annualized relapse rate, the two primary endpoints, asshown in Table 2. These significant effects were confirmed versusAVONEX® alone.

TABLE 2 Efficacy of Natalizumab in Phase 3 Studies Add-on therapyMonotherapy AVONEX + 300 mg AVONEX + 300 mg Placebo natalizumab placebonatalizumab Number of patients 315 627 582 589 Percentage of patientswith 29% 17% 29% 23% sustained progression of disability Hazard ratio(95% confidence interval) 0.58 (0.43, 0.77) 0.76 (0.61, 0.96) Riskreduction 42% 24% p-value p <0.001 p = 0.024 Annualized relapse rate0.733 0.235 0.749 0.336 Relative reduction 68% 55% p-value p <0.001 p<0.001

The patient population in the two Phase 3 studies were relapsing MSpatients according to the criteria of the International Panel on theDiagnosis of Multiple Sclerosis (McDonald et al., Ann. Neurol.50:121-127 (2001)). It encompassed a broad range of ages and diseaseseverity, and represented the current relapsing MS population withactive disease, consistent with the approved indication. Patients withprimary- or secondary-progressive MS were excluded.

The patient populations targeted for the two studies differed. Patientsin the monotherapy study were essentially naïve to treatment with animmunomodulatory drug for MS. Specifically, patients may not have hadtreatment with any immunomodulator (β-interferon or glatiramer acetate)for a period longer than six months and not within six months of thebeginning of the study. The result was a young, mostly female, MSpopulation with a moderate degree of baseline disease activity (typicalof the general MS population), very few of whom had tried anotherimmunomodulator prior to study entry.

Patients in the add-on therapy study were required to have receivedAVONEX® for the previous year and to have had a relapse during that timewhile on AVONEX® treatment. This resulted in a population somewhat olderthan that in the monotherapy study, with a longer disease duration.However, patients in the add-on therapy study had a similar degree ofdiseaseactivity as those in the monotherapy study, despite AVONEX®treatment.

1.-63. (canceled)
 64. A method of treating a patient with aninflammatory or autoimmune disease with an IgG4 anti-VLA-4 antibodycomprising: (a) detecting an amount of endogenous IgG4 in the patient'splasma or serum prior to initiating treatment; (b) determining a doseand dosage period of the IgG4 anti-VLA-4 antibody based on the amount ofIgG4 in the patient's plasma or serum; and (c) administering the dose ofthe IgG4 anti-VLA-4 antibody to the patient over the dosage period,wherein the dose and dosage period improve the safety and/or efficacy ofthe treatment compared to the safety and/or efficacy provided by thestandard dose and dosage period of the IgG4 anti-VLA-4 antibody.
 65. Themethod of claim 64, wherein the standard dose is 300 mg by IV infusionand the standard dosage period is every four weeks.
 66. The method ofclaim 64, wherein the amount of IgG4 in the patient's blood is below apredetermined level and the determined dose of IgG4 anti-VLA-4 antibodyis below the standard dose.
 67. The method of claim 64, wherein theamount of IgG4 in the patient's blood is below a predetermined level andthe administered dose of IgG4 anti-VLA-4 antibody is administered for adosage period longer than the standard dosage period.
 68. The method ofclaim 66, wherein the predetermined level is 200 μg/ml.
 69. The methodof claim 67, wherein the predetermined level is 200 μg/ml.
 70. Themethod of claim 64, wherein the amount of IgG4 in the patient's blood isbelow 200 μg/ml and the determined dose of IgG4 anti-VLA-4 antibody isbelow 300 mg by IV infusion.
 71. The method of claim 64, wherein theamount of IgG4 in the patient's blood is below 200 μg/ml and theadministered dose of IgG4 anti-VLA-4 antibody is administered for adosage period longer than four weeks.
 72. The method of claim 64,further comprising monitoring the patient for indicators of seriousinfection and/or treating the patient with prophylaxis designed toreduce the risk of developing serious infection.
 73. The method of claim64, further comprising monitoring the patient for indications ofprogressive multifocal leukoencephalopathy.
 74. The method of claim 73,wherein the monitoring detects JCV in the patient's urine, blood, and/orcerebrospinal fluid.
 75. The method of claim 73, wherein the monitoringcomprises testing for clinical and/or radiologic symptoms of progressivemultifocal leukoencephalopathy.
 76. The method of claim 73, furthercomprising, in the presence of indicators of progressive multifocalleukoencephalopathy, providing at least one treatment selected fromintravenous immunoglobulin therapy, plasmapheresis, and antiviraltherapy.
 77. A method of treating a patient with an inflammatory orautoimmune disease with an IgG4 anti-VLA-4 antibody comprising: (a)detecting an amount of endogenous IgG4 in the patient's plasma or serumprior to initiating treatment; (b) administering a dose of IgG4anti-VLA-4 antibody for a first dosage period; (c) monitoring a level ofbivalent IgG4 anti-VLA-4 antibody in the patient's plasma or serumduring the first dosage period; (d) determining a second dose and dosageperiod of IgG4 anti-VLA-4 antibody based on the amount of endogenousIgG4 in the patient's plasma or serum and on the level of bivalent IgG4anti-VLA-4 antibody in the patient's plasma or serum; and (c)administering the second dose of the IgG4 anti-VLA-4 antibody to thepatient over the second dosage period, wherein the second dose anddosage period improve the safety and/or efficacy of the treatmentcompared to the safety and/or efficacy provided by the standard dose anddosage period of the IgG4 anti-VLA-4 antibody.
 78. The method of claim77, wherein the amount of endogenous IgG4 in the patient's blood isbelow a predetermined level and the determined second dose of IgG4anti-VLA-4 antibody is below the standard dose or the determined seconddosage period is longer than the standard dosage period.
 79. The methodof claim 78, wherein the predetermined level is 200 μg/ml.
 80. Themethod of claim 77, wherein the monitoring shows that the amount ofbivalent IgG4 anti-VLA-4 antibody in the patient's blood is above apredetermined level during the first dosage period, and the administeredsecond dose of IgG4 anti-VLA-4 antibody is administered for a seconddose or dosage period that achieves a reduction of the amount ofbivalent IgG4 anti-VLA-4 to below the predetermined level during atleast a portion of the second dosage period.
 81. The method of claim 80,wherein the predetermined level is 1 μg/ml.
 82. The method of claim 80,wherein the determined second dose of IgG4 anti-VLA-4 antibody is belowthe standard dose or the determined second dosage period is longer thanthe standard dosage period.